
Class T ^G^U 
Book .7 ft> < | 

Copyright N°. 



COPYRIGHT DEPOSIT. 



Fig. 1. — A typical field seen by the in-vitro method of staining, 
cytes are staining gradually. 



The leuco- 



INDUCED CELL-EEPEO- 
DUCTION AND CANGEE 

THE ISOLATION OF THE CHEMICAL CAUSES OF NORMAL AND 
OF AUGMENTED, ASYMMETRICAL HUMAN CELL-DIVISION 



BY HUGH CAMPBELL ROSS 

M.R.C.S. (ENG.), L.R.C.P. (LOXD.) 

SURGEON, ROYAL NAVY (EMERGENCY LIST) ; DIRECTOR OF SPECIAL RESEARCHES AT THE 

ROYAL SOUTHERN HOSPITAL, LIVERPOOL; AND HONORARY CLINICAL PATHOLOGIST 

TO THE ROYAL LIVERPOOL COUNTRY HOSPITAL FOR CHILDREN 



BEING THE RESULTS OF RESEARCHES CARRIED 
OUT BY THE AUTHOR WITH THE ASSISTANCE OF 



JOHN WESTRAY CROPPER 

M.B., M.Sc. (LIV.), M.R.C.S. (ENG 1 .), L.R.C.P. (LOND.) 

ASSISTANT TO THE RESEARCH DEPARTMENT OF THE ROYAL SOUTHERN HOSPITAL, LIVERPOOL 



WITH 129 ILLUSTRATIONS 



PHILADELPHIA 
BLAKISTON'S SON & CO. 

1012 WALNUT STREET 
1911 



-&%\ 



Copyright, 1910, by P. Blakiston's Son & Co. 



Printed by 

The Maple Press 

York, Pa. 



CCLA27S555 



INSCRIBED TO 

SIR WILLIAM P. HARTLEY, J. P. 

OF LIVERPOOL 
AND 

JOHN HOWARD McFADDEN, Esq. 

OF PHILADELPHIA 




1 



A 



Mitotic Figure Induced in a Large Lymphocyte. 

This illustration was obtained after the book had gone to press and does not appear in 
the list of illustrations. It is included because it so clearly demonstrates the mitosis of the 
lymphocyte which was unstained. The division was induced by means of bensamidine, one 
of the several compounds containing the amidine grouping (N = C — N) the presence of which 
appears to be necessary in a substance before it can cause cell-division. This point was de- 
termined after the book had gone to press. 



PREFACE 



The objects of this book are to describe in detail the 
results obtained by a new method of experimentation 
with individual living human cells, their importance in 
the elucidation of the phenomena of healing, and in the 
causation of cancer and other growths. 

The old methods of examining dead tissues and 
cells have been useful in the past, but I venture to 
think that those who undertake the study of living 
human cells, and especially blood-cells, by the in-vitro 
methods of staining, which will be hereafter described, 
will realise that they supersede all others. This method 
enables us to observe cells in their proper shapes, and 
an entirely new impression is obtained concerning 
the functions of their constituent elements and of the 
modes by which they divide and reproduce themselves. 
The fact that the divisions of reproduction can be 
induced on a microscope slide by means of the natural 
chemical agencies which cause their proliferation within 
the body has in itself opened a fresh vista of research 
which has not only taught us the cause of prolifera- 
tion of cells in healing, but has also suggested that 
the cause of malignancy, which appears to be re- 
lated to that normal process, is beginning to become 
cleared up. 

The methods which I shall describe are entirely 

ix 



X PREFACE 

new; some of the details have already been published 
in the scientific journals, but the greater part of what is 
herein set forth has hitherto been unknown. The new 
methods have revealed many interesting facts which in 
my opinion may be far-reaching in their influence in 
the advancement of pathology. 

It may not be out of place if I give a brief history 
of the circumstances which led to the adoption of this 
in-vitro method of microscopical investigation and of 
the researches which have been made by means of it. 
There can be no doubt that accidents have on more 
than one occasion been responsible for valuable indica- 
tions which have led to fruitful lines of work, and had 
it not been for some of these accidents the results 
attained would have been considerably less advanced 
than they are now. I do not think that these re- 
searches would have been started at all had it not been 
for the firing of a gun. In the summer of 1905, when 
I was a surgeon in the navy, my cabin being my 
laboratory, I was interested in bacteriology, and 
was endeavouring to grow organisms from the blood 
of patients. To do this I had to invent an electric 
incubator, it being impossible on board a battle- 
ship to use one which was heated either by gas or oil, 
the former not being available and the latter not 
allowed. There was nothing for it, therefore, but to 
invent an incubator which could be made on board, 
and I look back upon this piece of apparatus with 
interest. It was not so reliable as those which can 
now be bought, but it worked fairly well. It had an 
automatic thermostat, by means of which the lamp was 



PREFACE XI 

switched out at a given temperature, and was switched 
on again when the temperature fell. Sparking gave 
trouble, but I "blew out" the spark by a condenser. 
It was made by a "torpedo instructor," and was so 
firmly bolted on to the steel bulkhead in my cabin that 
apparently nothing would (or could) shake it down. 

One afternoon, when my ship was in the Mediter- 
ranean, we had, as I thought, finished heavy gun-firing. 
I had placed some blood on to some nutrient agar 
(sloped) in culture-tubes, which were in my incubator, 
being kept at the blood temperature of 37° C. I was 
working at the cabin table with the microscope and my 
small stock of bacteriological apparatus. Suddenly, 
without any warning, a "young gentleman" fired a 
12-inch gun from the after-babette on the deck above; 
for the captain had permitted the midshipmen to fire 
a "round" after the main gunnery practice was over. 
I extricated myself from the debris of microscope, 
apparatus, pictures, etc., on the deck of my cabin, for 
nearly everything was smashed. My incubator, firmly 
fixed, as I have explained, on to the bulkhead, I did not 
open, expecting that everything inside was shattered, 
and it was not until the next day that I investigated its 
contents. My surprise may be imagined when I found 
that the culture-tubes were unharmed, but that, owing" 
to the dislocation of the automatic thermostat, the 
temperature inside the apparatus was standing at 60° C. 
On close examination of the culture-tubes, I noticed 
that the red cells, which had been resting on the surface 
of the jelly, were now diffusing as a cloud through the 
jelly itself. The matter was further investigated, and it 



Xll PREFACE 

formed the subject of my paper in The British Medical 
Journal of May 5, 1906, on "The Diffusion of Red 
Blood-corpuscles through Solid Nutrient Agar." The 
reason why the diffusion had not been previously 
observed was that one does not usually endeavour to 
obtain cultures from the blood at 60° C, nor should 
I have done so had it not been for the zeal of the 
"young gentleman." 

In January, 1906, before my paper was published, I 
was demonstrating this remarkable diffusion of red cells 
through the agar to my brother, Professor Ronald Ross, 
at Liverpool, by placing some blood under a cover-glass 
on a film of agar jelly spread on a slide. He was 
impressed by the way in which the cells became spread 
out between the cover-glass and the surface of the agar 
film, and he suggested that it would be a good means 
for blood-examination by the microscope, for the 
corpuscles became admirably arranged and spread out in 
such a way that each could be critically examined. 
Then he remarked — a remark which has led to all the 
researches described in this book, and to the discovery 
that mitotic divisions in human cells are induced by 
chemical agents — "I wonder what would happen if we 
were to mix some stain with the jelly and then place 
the living cells on it under a cover-glass." This sugges- 
tion was promptly put into operation, and fortunately 
(because it was the best which could have been chosen) 
the first stain experimented with happened to be poly- 
chrome methylene blue, with which I obtained results 
which determined me to adopt this method of examina- 
tion to the exclusion of all others. 



PREFACE Xlll 

In July, 1906, I left the navy and proceeded to 
Egypt, having received an appointment there in the 
Public Health Department. Sir Horace Pinching, 
the Director- General, permitted me to continue the 
researches, and it was during the ensuing year that 
the phenomena of achromasia and liquefaction of the 
cytoplasm of leucocytes were investigated together with 
some of the laws concerning the diffusion of substances 
into cells. In January, 1907, I accidentally discovered 
the excitation of amoeboid movements caused by 
atropine, for, as will be described in the chapter relative 
to this phenomenon, I was in reality trying to poison 
the cells with the alkaloid. 

In October, 1907, Sir Horace Pinching, my chief, 
having retired, I was treated in such a manner by 
Mr. W. P. Graham, the new Director-General of the 
Public Health Department, that I was forced to leave 
the Egyptian Government Service in December, 1907. 
Mr. Graham objected to my doing scientific work dur- 
ing my spare time, and also prevented my continuing 
the mosquito campaigns which I had started, as he 
apparently did not believe in them. This treatment 
stopped may researches for the time being, and was the 
cause of considerable delay in accomplishment of this 
work. I was enabled, however, to complete my investi- 
gations into the cause of achromasia by Dr. Marc 
Armond Ruffer, C. M. G., who came to my rescue and 
temporarily gave me an appointment in the Quarantine 
Department at Suakim in February, 1908. Here I 
was also able to devise the technics for " measuring the 
lives of leucocytes." 



XIV PREFACE 



In July, 1908, I obtained the appointment of Patho- 
logist to the Royal Southern Hospital at Liverpool, 
which I held for eight months, during which I was 
able to investigate further the laws of the diffusion of 
substances into living cells and to devise the technics 
for the determination of the "coefficient of diffusion." 
In the meantime I published the results of work done 
while I was in Egypt. This could not be done before, 
because Mr. Graham would not permit me to publish 
scientific work. Two papers appeared in The Journal 
of Physiology in September, 1908, four in The .Lancet 
in January and February, 1909, and that on the "Co- 
efficient of Diffusion" in the Proceedings of the Royal 
Society in April, 1909. 

In August, 1908, I was demonstrating the excita- 
tion of amoeboid movements caused by atropine to 
Dr. Macalister, when he suggested to me that possibly 
there might be some alkaloid-like excitant in the blood 
of cancer patients; and this important suggestion was 
the starting-point of the investigation of cancer by this 
in-vitro method. Dr. Macalister and I read a paper 
before the Royal Society of Medicine in November, 
1909, on the researches which immediately followed his 
suggestion. 

In March, 1909, Professor Harvey Gibson also sug- 
gested the important point, based on an observation 
made by Professor Farmer, that nuclein might have 
some influence on cell-division. I must acknowledge 
the great assistance which I have received from Pro- 
fessor Harvey Gibson on many occasions, as well as 
the loan of a well-equipped laboratory in the Hartley 



PREFACE XV 



Botanical Department at the University of Liverpool. 

It was when experimenting with a mixture of 
polychrome stain, extract of haemal gland, and atropine 
that I saw mitotic figures in lymphocytes for the first 
time in May, 1909; and the ensuing months were 
occupied in the investigation of the cytology of these 
cells and of the means whereby they might be induced 
to reproduce themselves. It was not until October, 
1909, however, that I was able to induce divisions in 
polymorphonuclear leucocytes. In December, 1909, I 
discovered almost accidentally that extracts of dead 
tissues, if they were allowed to decompose by the 
action of putrefactive bacteria, would, by themselves, 
induce the division and multiplication of lymphocytes, 
and this was followed by the investigation of the 
action of "globin" in January, 1910. In February, 1910, 
while investigating the epithelial cells present in some 
vaginal secretion, it occurred to me to try to induce 
divisions in them, and this experiment has been suc- 
cessful in the case of one or two cells. 

In April and May, 1910, when working with my 
assistant, Dr. Cropper, I saw divisions induced by 
kreatin and xanthin, the "extractives" contained in the 
remains of dead tissues; and we then also investigated 
the augmenting action on cell-division of the alkaloids 
choline, cadaverine, etc., produced by the decompo- 
sition of putrefaction. These points led me to elaborate 
the theory regarding the cause of cancer which is 
described in the latter part of this book. 

The dates of the treatment of the two cases of 
cancer are given in the description of them which 



XVI PREFACE 

has been written by Dr. Macalister. The crucial 
experiment to try to determine whether my theory 
regarding the cause of cancer was correct or not was 
made in the first week of August, 1910. The treatment 
of all the cases has been carried out under the immediate 
supervision of Dr. Macalister. 

In much of the latter portion of the experimental 
work I have derived assistance from Dr. Cropper. He 
has accomplished nearly all the investigations con- 
cerned in counting the number of granules contained 
in eosinophile leucocytes, and has given most valuable 
assistance in the isolation of the active "auxetics" 
from the remains of dead tissues, and in the investiga- 
tion of the inhibitory action of blood-serum, some of 
which he did entirely himself at my suggestion. 

The Research Department at the Royal Southern 
Hospital at Liverpool was started by Dr. Macalister 
in April, 1909, in order that this work might continue; 
Sir William Hartley, J. P., generously supplied funds 
to last for one year in the first instance. In November, 
1909, he extended his support for a period of three 
years, and he also supplied me with the photomicro- 
graphic camera which I had invented. Some of the 
expenses attached to these researches, however, have 
also been defrayed by Mrs. George Holt, Mr. and the 
Misses Paton, and some of their friends. I would 
like to take this opportunity of recording my personal 
gratitude to Sir William Hartley and these ladies and 
gentlemen, without whose assistance these researches 
could not have been accomplished. 

I wish also to record the manner in which I was 



PREFACE XV11 



enabled to obtain the assistance of Dr. Cropper. In 
October, 1909, I happened to be discussing with Mr. 
Sharpies, an energetic supporter of these researches, 
the difficulty of my finding time to undertake the 
investigation of some of the by-issues revealed by the 
new method — issues which might prove to be of 
importance. Our conversation was overheard by a 
gentleman, who I afterwards ascertained was Mr. 
J. H. McFadden, of Philadelphia, whose acquaintance 
I had only just made, and to whom I was practically 
a stranger. Mr. McFadden immediately became in- 
terested, and placed a large sum of money at my 
disposal in order that I might obtain other assistance 
to further these researches for a period of two years. 
In March, 1910, Mr. McFadden further instructed me 
to the effect that if I conscientiously thought that 
further funds could usefully be spent in the advance- 
ment of these researches I was to incur that expendi- 
ture. In fact, he has not only supplied me with the 
assistance of Dr. Cropper, but he has also been the 
means of equipping a laboratory for him, kindly lent 
by the Liverpool School of Tropical Medicine, but 
also in defraying the serious expenditure connected 
with the manufacture of the substance we call "globin" 
from crystalline haemoglobin. Mr. McFadden has also 
enabled me to take the large number of photomicro- 
graphs which record the phenomena seen under the 
microscope; and, lastly, he has borne the entire cost 
of the publication of this volume and the reproduction 
of the photomicrographs which illustrate it. 

I fear that I shall never be able to thank Mr. 



XV111 PREFACE 

McFadden sufficiently for his great generosity, which 
I appreciate very greatly. It was extended to me at 
a time when I was practically a total stranger to 
him, together with the intimation that even if these 
researches did not result in the advancement of knowl- 
edge regarding cancer he would not consider that his 
assistance had been misplaced or wasted. 

In conducting prolonged researches of this nature 
it is most gratifying to realise that one has such a 
staunch supporter; and there can be no question that 
if the results obtained lead to practical benefit, this will 
be largely owing to Sir William Hartley, who enabled 
the researches to be started, and to Mr. McFadden, 
who enabled them to be brought so rapidly to the point 
which has now been reached. 

When this Research Department of the Royal 
Southern Hospital was started, a Committee was 
formed. It numbers amongst its members Professors 
Sherrington, Herdman, Ronald Ross, Reynolds Green, 
and Harvey Gibson, to all of whom I have frequently 
appealed for advice on technical points; and when any 
information has been required concerning the surgical 
aspects of the healing process, or of cancer, I have 
consulted Mr. Robert Jones, who is now Chairman of 
the Committee, and also Dr. Alexander. I wish to 
thank all these gentlemen most sincerely for their 
kindness. I have also frequently received materials 
from Mr. Jeans and Mr. Bickersteth, of the Royal 
Infirmary, and many other members of the medical 
profession in Liverpool have supplied specimens. The 
beautiful sections of the growth with which the crucial 



PREFACE XIX 

experiments were made were kindly cut for me by 
Dr. Moore Alexander. Professor Benjamin Moore 
advised us in our researches to isolate the active auxetics 
from the extracts of dead tissues, and Dr. H. E. Roaf 
very kindly supplied us with the crystalline haemoglobin 
from which globin was obtained for the first ex- 
periments with that substance. 

I cannot close this Preface without signifying my 
thanks to Dr. Macalister. He grasped that this in- 
vitro method would bring fruitful results, and it was 
he who instituted this research into the cause of 
cancer. Dr. Macalister's advice and constant encour- 
agement, apart from the actual experimental work 
which he has done and the clinical observations which 
he has made, have been invaluable. 



CONTENTS 



CHAPTER I 



PAGE 

The Scope of the New Method 1 



CHAFTER II 

The General Principles of the Method — The Appara- 
tus required — The Special Photomicrographic 
Apparatus — The Revolving Apparatus 15 



CHAPTER III 

The Preparation of the Jelly-film 36 

CHAPTER IV 

Cellular Staining, Death, and Achroal\sia 41 

CHAPTER V 

The Diffusion of Substances into Living Cells — The 

" Coefficient of Diffusion" 61 

CHAPTER VI 

The Practical Determination of the "Coefficient of 
Diffusion" of Cells, and its Application to this 

"in-vitro" Method of Research 81 

xxi 



XX11 CONTENTS 

CHAPTER VII 

PAGE 

Diffusion of Substances into Cells to excess — Diffu- 
sion- vacuoles or " Red Spots" — The Proofthat the 
Blood-platelet is a Living Cell 103 

CHAPTER VIII 

The Excitation of Amoeboid Movements in White 

Blood-corpuscles caused by Alkaloids 130 

CHAPTER IX 

The Adoption of the "in- vitro" Method for Cancer 
Research — The Excitation of Leucocytes caused 
by Cancer Plasma — Facts known about Cancer — 
The Age-incidence; Vitality; Death; Metastasis; 
Chronic Irritation — The Possible Causes of Cell- 
proliferation discussed 157 

CHAPTER X 

Experiments with Nuclein — The Lowering of the Co- 
efficient of Diffusion caused by Extracts of Dead 
Haemal Gland — Divisions induced in Lymphocytes 
for the First Time — Revelations concerning these 
Divisions — The Roles played by the Altmann's Gra- 
nules, Nuclei, and Nucleoli in the Cell-division 172 

CHAPTER XI 

The Division of Lymphocytes induced by the Aniline 
Dye — The Augmenting Action of Atropine and 
Extract of H^mal Gland — "Auxetics" — The Cycle 
of Cell-division — The Possibilities of the Induced 
Cell-division being due to "Death-struggles" — 
Asymmetrical and Reduced Divisions 225 



CONTENTS XX111 

CHAPTER XII 

PAGE 

The "Experimental Ten Minutes" — Division induced 

in the so-called polynuclear leucocytes method 

for Counting the Number of Granules in Eosino- 
phile Leucocytes, and the Reduction of this Num- 
ber in the Cells of Cancer Patients 249 

CHAPTER XIII 

The Auxetic Action of Cancer-serum — The Induced 
Divisions of Granular Red Cells — The Auxetic 
Action of "the Remains of Dead Tissues," and its 
Augmentation by Atropine and the Products of 
Putrefaction — The Isolation of the Auxetics 
Kreatin and Xanthin — Discovery of Causes of the 
Cell-proliferation of Healing 292 

CHAPTER XIV 

The Auxetic Action of Globin 



CHAPTER XV 

The Proof that the Remains of Dead Tissues and Glo- 
bin CONTAIN THE CAUSES OF THE CeLL-PROLIFERATION 

of Healing, and other Cell-reproduction — Experi- 
mentation "in vivo" confirms "in-vitro" Observa- 
tions — The Cause of Benign Tumours 333 

CHAPTER XVI 

The Augmented Divisions induced by Putrefaction 
of the Extracts is due to the Alkaloids of Putre- 
faction — A Theory that Carcinoma and Lympha- 
denoma may be caused by the Combination of the 
Auxetics of Cell-proliferation and Choline and 
Cadaverine — An Explanation of the Age-incidence, 
Metastasis, and the other Facts known concerning 
Cancer — The Necessity for a Crucial Experiment 
to prove the theory 348 



XXIV CONTENTS 

CHAPTER XVII 

PAGE 

The Inhibitory Action of Blood-serum in preventing 
the Action of Auxetics in causing Cell-division — 
Measurement of this Action — The Treatment of 
some Cases of Cancer by Defibrinated Blood — 
Description of the Cases — The Treatment of a 

Malignant Ulcer by means of Globin — The Crucial 

Experiment — Conclusion 374 

APPENDIX I 

Tables describing the Enumeration of the Number 

of Granules contained in Eosinophile Leucocytes 401 

APPENDIX II 

Method for estimating the Number ol Living and Dead 
Leucocytes contained in a given Sample of Blood, 
and measuring the llves of leucocytes 406 

APPENDIX III 

A "Hanging-drop" Preparation with the Jelly Method 419 

APPENDIX IV 

A Contribution to the " Theory of Immunity" 420 

INDEX 421 



LIST OF ILLUSTRATIONS 



All the photomicrographs which illustrate this book were taken 
with the apparatus described in Chapter II. The objective used for 
those taken with the higher magnification was a 2-mm. apochromatie 
lens (Zeiss) N. A. 1*30. The objectives employed for taking photo- 
graphs of a lower magnification were Zeiss D and Zeiss A. The eye- 
piece used in all of them was a "high-power projection eye-piece"" 
(Watson). 

In the actual preparations, as observed through the microscope, a 
stereoscopic view of the dividing cells can be obtained, which facilitates 
the demonstration of the different phases. Unfortunately, this stereo- 
scopic effect cannot be seen in the prints, although an examination of 
them with a hand magnifying-glass will remedy the deficiency to some 
extent. 

The photograghs have all been produced without any alteration of 
the original negatives. 

FIG. PAGE. 

1. A typical field seen by the in-vitro method of staining. The leuco- 

cytes are staining gradually Frontispiece. 

2. The photomicrographic apparatus. The microscope is ready to 

be used for direct observation. The gas-burner can just be seen 
at the lower end of the wooden plank. (N. B. — The sheets of 
white paper have been placed in this position in this and the next 
photograph in order to "show up" the apparatus.) 23- 

3. The apparatus ready for photography. The mirror is swung aside, 

and the eye-piece attached to the camera is inserted into the 
microscope 25- 

4. The photomicrographic apparatus. Showing positions of water- 

cooling tank and Nernst burner. The microscope mirror is in 
position for direct observation 29' 

5. The photomicrographic apparatus. The microscope mirror is 

swung aside for photography 31 

6. The granules of the leucocyte are gradually becoming stained. 

The red cells are unstained. Low power 45- 

7. The leucocyte's granules are stained. Its nucleus is unstained. 

The pseudopodia are extruded in response to atropine, which is 
diffusing into the cell as well as the stain 4S 

XXV 



XXVI LIST OF ILLUSTRATIONS 

FIG- PAGE. 

8. The same field as 7. The leucocyte is retracting its pseudopodia. 47 

9. The same field as 7 and 8. The retraction of pseudopodia is nearly 

complete. The lobes of the nucleus of the leucocyte are turning 

a faint blue colour 47 

10. A leucocyte excited by atropine. Its granules are deeply stained, 

and its nucleus is also beginning to stain a blue colour. Low 
power 49 

11. A leucocyte which has j ust been killed by the staining of its nucleus. 

Its granules are also deeply stained 49 

12. The leucocyte has just died owing to the staining of its nucleus. 

The cell-wall is beginning to bulge because the cytoplasm is 
liquefying 53 

13. The onset of achromasia. The same field as 12. The stain is 

beginning to fade from the nucleus. The bulging of the cell- 
wall has become general 53 

14. Achromasia. The same field as 13. The stain has gone from the 

nucleus, although the granules are still stained. Note that the 
red cell is disappearing 57 

15. Achromasia. The same field as 14. Many of the cell-granules 

have lost their stain. The cell- wall is nearly invisible. The 
red cell has disappeared 57 

16. A stained leucocyte. The ordinary vacuoles (colourless patches 

amongst the cell granules) are well shown. The cell has just 
died 105 

17. Diffusion- vacuoles in a leucocyte 105 

18. A dead leucocyte in which diffusion-vacuoles are beginning to 

appear 109 

19. A diffusion-vacuole in a lymphocyte. Low power 109 

20. A diffusion-vacuole in a granular red cell 115 

21. A clump of normal blood-platelets. They are resting on a jelly 

which will just stain their granules 115 

22. Diffusion-vacuoles in blood-platelets. The cells are resting on the 

same jelly-film as those in 21, but they had been subjected to 
the action of morphine hydrochloride 119 

23. Diffusion-vacuoles in blood-platelets. The jelly-film had the 

same index of diffusion as that employed in 21 119 

24. A specimen of blood which had been mixed with morphia solution. 

Note the extreme vacuolation of the leucocyte. A blood- 
platelet is also vacuolated. The same jelly as in 21 121 

25. Patches resembling archoplasm induced in a leucocyte by sub- 

jecting the blood to an extract of a dead tissue. The jelly-film 

on which the cells are resting is similar to that employed in 21 . 121 

26. An extruded pseudopodium becoming detached from a leucocyte 

which is excited by atropine. No stain 125 

27. Amoeboid movements excited in a blood-platelet by the action of 

atropine 125 



LIST OF ILLUSTRATIONS XXV11 

FIG. PAGE. 

28. Amoeboid movements excited in a leucocyte by the action of 

atropine. Low power 135 

29. Exaggerated amoeboid movements in leucocytes which have their 

granules stained. The movements were excited by atropine 
sulphate 135 

30. Excited leucocytes extruding their pseudopodia between red cells . 137 

31. Excitation of amoeboid movements in a lymphocyte by the action 

of atropine. No stain 137 

32. Excitation of amoeboid movements in a lymphocyte which has its 

granules stained 143 

33. Extreme excitation of amoeboid movements in a lymphocyte. 

No stain 143 

34. Excitation of two leucocytes by the action of choline. Low 

power. No stain 151 

35. Excitation of a lymphocyte by the action of choline. No stain . 151 

36. Excitation of amoeboid movements in a leucocyte by the action of 

cadaverine. No stain 153 

37. A leucocyte excited by morphine. The cell's granules are stained . 153 

38. Leucocytes excited by pyridine. No stain 173 

39. A lymphocyte which has absorbed stain and atropine discarding 

its granules (flagellation) 173 

40. A resting lymphocyte. Note the deeply stained masses of gran- 

ules in the cytoplasm, which is bulged out in places. The large 
transparent nucleus and the stained ring-shaped nucleolus can 
also be seen 189 

41. A resting lymphocyte. The Altmann's granules in the cytoplasm 

are stained 189 

42. A resting lymphocyte. The cytoplasm, the granules, the nucleus, 

and the nucleolus can be distinguished 191 

43. The earliest stage of mitosis. The nucleolus has divided into two 

rings 191 

44. Early mitosis in a lymphocyte. Looking down through the spindle 

(polar aspect) . The nucleolus has divided into two centrosomes, 
each of which is ring-shaped. The spindle is surrounded by a 
belt of chromatin granules 193 

45. Mitosis in a lymphocyte. Profile aspect. The two ring-shaped 

centrosomes can just be seen towards the poles. The granules 
are becoming formed into chromosomes 193 

46. Foreshortened appearance of a mitotic figure in a lymphocyte. 

The position of one nucleolus-centrosome at the pole of the figure 

is well shown 195 

47. Profile aspect of mitosis in a lymphocyte. The relative positions 

of the centrosomes and chromosomes can be seen 195 

4S. Profile aspect of mitosis. The belt of chromatin is formed round 

the waist of the cell 197 



XXV111 LIST OF ILLUSTRATIONS 



PAGE. 



49. One resting and one dividing lymphocyte. In the latter the 

chromosomes are beginning to divide. The centrosomes appear 

as dots of chromatin "... 197 

50. Polar aspect. The belt of chromatin granules is dividing into 

chromosomes 199 

51. Polar aspect. The chromosomes are becoming semicircular . . 199 

52. Polar aspect. An " aster " stage of mitosis in a lymphocyte . . 201 

53. Polar aspect. Some of the chromosomes are semicircular-shaped; 

some are dots of chromatin 201 

54. Polar aspect. One centrosome can be seen at the pole of the 

"aster" figure 203 

55. Polar aspect. Sixteen chromosomes could be counted in this cell. 203 

56. Profile aspect, of mitosis in a lymphocyte 205 

57. Profile aspect of mitosis in a lymphocyte 205 

58. Profile aspect. The chromosomes can be seen at the waist of the 

spindle 207 

59. Profile aspect. A figure frequently seen 207 

60. Profile aspect of mitosis 209 

•61. Oblique aspect of mitosis in a lymphocyte 209 

62. Polar aspect of mitosis in a large lymphocyte from a patient 

suffering from carcinoma. There are sixteen chromosomes . .211 

63. Polar aspect. The chromosomes were V-shaped with their apices 

inward to be attached to the nucleus-spindle, which can dimly 

be made out 211 

64. Polar aspect of mitosis in a large lymphocyte from a cancer 

patient. The chromosomes are dividing 213 

65. Profile aspect of mitosis 213 

66. Profile aspect. The figure is fully formed. One nucleolus-centro- 

some is ring-shaped; the other is a dot of chromatin 215 

67. Profile aspect. The sixteen chromosomes could be counted . . 215 

68. The cell has become constricted in its centre 217 

69. Profile aspect. Complete division is about to occur. The chro- 

mosomes are being reconverted into granules, but the mitotic 
figure is not quite finished at the dividing-point 217 

70. Profile aspect. The spindle and chromosomes have divided, but 

the cell- wall has not yet separated 219 

71. Completion of mitosis in a lymphocyte 219 

72. Asymmetrical mitosis in a lymphocyte induced by azur stain aug- 

mented by atropine 233 

73. Asymmetrical mitosis induced by azur stain augmented by 

atropine 233 

74. An early stage of delayed mitosis induced by a jelly with a low 

index of diffusion. The number of chromosomes is more than 
sixteen 241 

75. Thirty-two chromosomes could be counted in this cell. Early 

mitosis delayed 241 



LIST OF ILLUSTRATIONS XXIX 

FIG. PAGE. 

76. A resting polymorphonuclear leucocyte. Its granules are stained 

but not its nucleus. The cell was alive 253 

77. A basophile leucocyte in the act of cell-division. The granules 

of the cell are in the centre. The lobes of the nucleus are at the 
poles of the cell which is dividing into three 253 

78. An eosinophile leucocyte in the earliest stage of division. The 

granules were arranged in lines radiating outwards from the 
centre of the cell. The lobes of the nucleus were at the poles . . 259 

79. Early stage of division of a neutrophile leucocyte 259 

80. A dividing leucocyte 261 

81. A dividing leucocyte 261 

81a. Division of a leucocyte. The linear arrangement of the granules 

could be well seen 263 

82. A dividing leucocyte 263 

83. A dividing leucocyte 265 

84. A dividing leucocyte 265 

85. A dividing leucocyte 267 

86. A dividing leucocyte 267 

87. An eosinophile leucocyte with its granules stained 275 

88. A field containing a neutrophile, an eosinophile, and a basophile 

leucocyte. The upper cell is the neutrophile and the lower one 
the basophile cell. All the cells are ruptured, but their granules 
are stained 279 

89. A basophile leucocyte whose stained granules have been turned 

black by heat 279 

90. One of the negatives of a ruptured eosinophile leucocyte (negative 

Xo. 52) 285 

91. One of the negatives of a ruptured eosinophile leucocyte (negative 

Xo. 54) 285 

92. Counting the granules. The image of the ruptured cell depicted 

on negative Xo. 52 is projected on to a sheet of white paper 
pinned on to a screen 287 

93. Counting the granules of negative Xo. 54 287 

94. A dividing red cell from a cancer patient 295 

95. A dividing red cell from a cancer patient. The granules seem to 

be arranged in an indefinite figure 295 

96. Very early stage of mitosis in a lymphocyte induced by decom- 

posed extract of suprarenal gland. Xo stain 301 

97. Mitosis of a lymphocyte induced by decomposed suprarenal 

extract. Xo stain 301 

98. Mitosis induced in a lymphocyte by decomposed extract. Xo 

stain 303 

99. Asymmetrical division induced by decomposed extract. Xo 

stain or atropine is present 303 

100. Mitosis induced by fresh extract of suprarenal gland. Xo stain 

or augmentor present 307 



XXX LIST OF ILLUSTRATIONS 

FIG. PAGE 

101. Mitosis induced by fresh suprarenal extract. No stain is present. 307 

102. A dividing polymorphonuclear leucocyte induced by suprarenal 

extract alone. No stain 311 

103. Mitosis induced in a lymphocyte by suprarenal extract which had 

purposely been allowed to become putrid. No stain . . . .311 

104. Mitosis induced in a lymphocyte by suprarenal extract which had 

purposely been allowed to become putrid. No stain .... 313 

105. Asymmetrical mitosis induced by suprarenal extract augmented 

by atropine. No stain 313 

106. Mitosis induced in a lymphocyte by kreatin. No stain or extract . 317 

107. Division in a leucocyte induced by kreatin. No stain or extract . 317 

108. Mitosis in a lymphocyte induced by globin augmented by atropine. 

No stain, extract, or kreatin 327 

109. Asymmetrical mitosis induced by globin augmented by atropine. 

No stain, extract, or kreatin 327 

110. Mitosis induced in a lymphocyte by means of decomposed globin 

solution. No stain, extract, kreatin, atropine 329 

111. To show the way in which globin is " dotted " over the sufrace of an 

ulcer 343 

112. To show the scab formed by the application of globin to an ulcer. 343 

113. Mitosis induced by a mixture of kreatin and choline. No stain, 

extract, or atropine 353 

114. Asymmetrical mitosis induced in a lymphocyte by a mixture of 

suprarenal extract and globin, augmented by choline. No 
stain or atropine 353 

115. Mitosis in a lymphocyte induced by globin and choline. No stain 

or other auxetic 355 

116. Mitosis induced in a lymphocyte by suprarenal extract and choline. 

No stain or other auxetic 355 

117. Mitosis induced in an epithelial cell by a mixture of stain and 

extract 357 

118. Early mitosis in an epithelial cell from the vagina induced by stain 

and extract 357 

119. Section from the case of scirrhus of the breast. Low power . . 385 

120. The same as 119. High power 385 

121. To show the way in which globin was "dotted" on to a portion of 

the malignant ulcer 387 

122. Section of a portion of the ulcer after treatment. Low power . . 391 

123. The same as 122. High power 391 

124. Section of the treated portion of the ulcer after the application of 

globin augmented by choline, showing reinfiltration. Low 
power 393 

125. The same as 124. High power 393 



Induced Cell- Reproduction 
and Cancer 

CHAPTER I 

THE SCOPE OF THE NEW METHOD 

The study of the individual living human cell and of 
the effects of chemical reagents upon it marks what 
may almost be regarded as a new scientific departure. 
Although much has been written concerning the 
passage of substances into cells, mainly the outcome 
of experiments not made actually upon the individual 
cells themselves, and certainly not while they were alive, 
little practical work has been done with reference to 
the behaviour of individual cells while substances are 
being made to pass into them. This has been owing 
to the lack of satisfactory methods, and because the 
laws which govern the diffusion of substances into the 
individual living cells have not been recognised. These 
laws are of the greatest importance, and must be 
thoroughly understood if in-vitro experimentation is 
to prove serviceable or successful, and later on a 
section will be devoted to this subject. In the mean- 
time the elemental fact must be simply stated that 
living cells are examined by placing them, under a 
cover-glass, on to the surface of a film of jelly, which 
may contain dissolved in it any substance we may wish 

1 



2 THE SCOPE OF THE NEW METHOD 

to experiment with, and which has, while in a molten 
condition, been poured on to a microscope slide and 
allowed to set there. The jelly may, for instance, 
contain an aniline dye; and by watching the way in 
which the living cells absorb the stain from the jelly, 
and by experimentation with it, many of the laws of 
the diffusion of substances into living cells have been 
ascertained; and by the application of these laws we 
can now add other substances to the jelly and make 
them also diffuse into the living cells, and watch the 
results by means of the microscope. The cells are 
pressed into the jelly by the cover-glass, and therefore 
they can absorb only what is in the jelly (there is 
nothing else for them to take), provided that the 
conditions have been correctly arranged for the pass- 
age of the substances from the jelly into the cells. It 
is essential to note that one class of cells differs from 
another with reference to the rate at which they absorb 
materials from the media in which they are placed, so 
that the composition of any given jelly must be cor- 
rectly arranged for experimentation with any particular 
class of cell with which it may be desired to work. 

The word "cell" in this book refers to the living 
cell unless otherwise specified. Cells must always be 
freshly removed from the body when they are placed 
on the jelly. It occasionally happens that the cells 
may have just died or be dying when they are ex- 
amined, as when mitotic divisions are being induced 
by azur stain, as will presently be described; but, 
generally speaking, after the cells in a specimen are 
dead the specimen is thrown away. It is obvious 



ADVANTAGES OF THE " IN- VITRO " METHOD 3 

that "specimens" of living cells cannot be kept. All 
attempts to "fix" the jelly films (on which the cells 
are resting) at the end of the experiments have so far 
failed, so it is impossible to retain the specimens for 
future examination or for purposes of collection; and 
consequently when dead, or when finished with, speci- 
mens have to be discarded. This circumstance has 
led me, at the suggestion of Professor Sherrington, to 
devise a rapid method of recording the actual experi- 
mental facts observed by means of photomicrography; 
and although the photographs, many of them taken with 
the highest powers of the microscope, are not com- 
parable by any means to what is seen with the eye, 
we at least have the satisfaction of knowing that a 
truthful image is recorded which cannot be influenced 
in the way that drawings, however carefully made, are 
apt to be. The photomicrograph is therefore the best 
substitute for microscope "specimens" which we have 
to offer. 

In the past very little has been learned from the 
study of individual living cells either in physiology or 
in pathology. Presumably this has been due to the 
fact that it has been difficult to stain cells satisfactorily 
when they are alive; for, since the discovery of the 
aniline dyes, stains have been used in nearly all micro- 
scopical work. It is true that a good deal of work 
has been done in the way of attempting to stain unfixed 
cells by mixing them with solutions of methylene blue 
and neutral red; but the results have not been very 
satisfactory, and no doubt the advances made in the 
study of dead cells by means of differential staining 



4 THE SCOPE OF THE NEW METHOD 

with dyes dissolved in alcohol have done something to 
retard in-vitro methods of investigation, because dyes 
dissolved in alcohol cannot, of course, be used to stain 
living cells. As a matter of fact, with this new "jelly" 
method it is simpler to stain certain living cells than it 
is to stain dead ones by the old methods, and better 
pictures are obtained although less skill is required. 
No matter how rapidly a cell or tissue is killed, the 
fact remains that it is dead, and the means usually 
taken to prepare it for examination by placing it in 
preservative solutions or in others necessary for fixing 
and staining it — not to speak of the processes of em- 
bedding and freezing and the subsequent cutting with 
razors and so forth— can only add to the fallacious 
results of its examination. So far as blood-cells are 
concerned, the study of their morphology and cystology 
has hitherto been almost entirely based on the exami- 
nation of dead specimens, with the result that some 
erroneous impressions, both as to form and func- 
tion, have become generally accepted. For instance, 
let an experienced worker with the older methods 
look for a "hyaline leucocyte" with the new one, and 
he will marvel at his credulity. The hyaline leuco- 
cyte is a dead lymphocyte which has become achro- 
matic. By the new method we see cells stained while 
they are alive, and admirably spread out on the jellies, 
so that they can readily be examined by the highest 
powers. One can now cause any soluble substance to 
diffuse into them at any rate one pleases, and with the 
help of this knowledge one can, by specific chemical 
agents, cause leucocytes and other cells to divide on 



IT REVEALS FALLACIES O 

the microscope slide. By this study of vital activity 
new lessons have been learned concerning the real 
functions of the morphological elements of the cells. 
For instance, owing to the fact that the older methods 
merely showed pictures of dead cells and the arrange- 
ment of their component parts after they are dead, 
controversies have arisen regarding the functions of 
the cellular elements. Unfortunately, theories regard- 
ing these functions have sometimes become accepted 
as facts. The "lobes of the nuclei" of leucocytes are 
generally recognized as being analogous to the nuclei 
of other cells, in spite of the fact that the act of cell- 
division has never been seen in leucocytes. 1 The very 
designation of the cells — "polymorphonuclear"— is 
even based on this theory; but in reality the "lobes 
of the nuclei" are the centrosomes. We hear it said 
even now that the blood-platelet is a precipitate, 
although a single glance at a specimen in vitro, espe- 
cially if an alkaloid is present in the jelly, demonstrates 
beyond denial that a blood-platelet is a living creature 
and a highly amoeboid cell. 

The new method reveals new points in every direc- 
tion which are difficult to reconcile with the old 
theories based upon the examination of dead specimens, 
some of them so firmly rooted that people may be slow 
to discard them. 

Infinite interest and variety awaits the investigator 
of cells by this new method. He is dealing with living 

1 Throughout this book the word ''leucocyte" refers to the polymorpho- 
nuclear cell; the mononuclear cell from the peripheral circulation is called a 
lymphocyte. 



6 THE SCOPE OF THE NEW METHOD 

creatures which are amenable and can be excited or 
made to divide almost at will. It is remarkable to 
think that one can order samples of one's own or some 
other person's white blood-corpuscles to reproduce 
themselves at a given time, and that if they are properly 
treated they will do so with obedient regularity. 
Instead of the diagrammatic representations of karyo- 
kinesis, from which every student learns his impressions 
of cell-division, one is now able to appreciate mitosis 
in its reality and to watch it through its various phases. 
This is a very striking fact, but its interest grows when 
we consider another very important lesson derived from 
it, insomuch that, as will be seen later, there is strong 
evidence that white corpuscles will multiply only in 
response to a specific chemical agent. We now believe 
that it is essential for a leucocyte to absorb an "aux- 
etic" (avi ^rtKo?, an exciter of reproduction) before it will 
make any attempt to proliferate, and we have also 
evidence that it is more than probable that other 
human cells, and possibly all of them, proliferate in 
response to a similar agency. It will be realized, there- 
fore, that this method of study of the cell and of the 
influences of chemical agencies upon it has opened 
up a new field of work, not only in pathology, but in 
physiology also. 

The proliferation of cells is the main theme of this 
book. By this in-vitro method it has not only been learnt 
that cells will divide in response to certain chemical 
agents, but that these agents exist in the remains of 
all dead tissues. Two of the substances which are 
directly responsible for cell-reproduction within the 



IT ELUCIDATES CELL-DIVISION i 

body have been isolated in crystalline form: they are 
the extractives, kreatin and xanthin, and individual 
cells divide in response to them according to the 
amount of each substance absorbed by the cell. 

It will be shown that cell-proliferation depends upon 
cell-death, and this affords an explanation of the cause 
and origin of benign tumours. "Development" is a 
basis of physiology; and since the multiplication of 
human cells is due to chemical agents, as is shown by 
this method, one cannot but suppose that the facts 
learnt may lead to the explanation of points connected 
with the growth of the embryo. 

One of the foundations of pathology is the phenome- 
non of "healing," which is caused primarily by the 
proliferation of certain cells. The causes of this 
proliferation have been ascertained for the first time 
by this method, and the ultimate chapters of this 
book will describe proofs that these causes are now 
known. If the finger is cut, or if disease gains a 
footing in any part of the body, an attempt is made 
by the tissue-cells to proliferate and to heal the injury; 
but up to now no one has known why this proliferation 
took place or how it was caused. This mystery is now 
elucidated. The knowledge that cell-proliferation in 
the body is due to chemical exciters, of reproduction 
(auxetics) is, we think, the beginning of an innovation 
which must lead to developments of practical value. 

The effect of any given substance, so long as it is 
soluble, can be tested on many individual human cells 
and the results watched. I fear that we, personally, 
have only been able so far to try the effects of auxetics, 



O THE SCOPE OF THE NEW METHOD 

alkaloids, and a few other substances ; but a whole field 
of investigation of the actions of substances on indi- 
vidual cells remains to be carried out, and this is now 
possible by this "jelly" method of in-vitro staining. 

The action of chemical substances on living cells 
is closely associated with the diffusion of substances 
into these cells (a subject to which a section of this 
book will be devoted), and this diffusion is governed 
by the "coefficient of diffusion" of the cells them- 
selves, a phenomenon which has been so far entirely 
studied by this in-vitro method. Up to the present, 
however, we have only had time to ascertain the com- 
parative rates of diffusion of substances into some of 
the classes of human cells and into a few species of 
bacteria. The determination of the coefficients of 
diffusion of all the rest of the cells of the whole ani- 
mal and vegetable kingdoms remains as a "legacy" 
for those who will undertake the work. 

Methods will be described by which the lengths of 
the lives of leucocytes can be measured after they 
have been removed from the body. By this means 
the comparative effects of different poisons on the 
cells can be tested, and the small amount of work 
done in this direction will be summarised. We think 
that there are possibilities that farther investigation 
of the actions of specific poisons, such as bacterial 
toxins, will lead to fruitful results; in fact, one of us 
(C. J. M.) has already shown by this method that 
chorea and rheumatism are less closely related than 
is generally supposed. 1 

1 British MedicalJournal, August 2S ; 1909. 



SHOWS THE CELL-STRUCTURE 9 

In the last chapter experimental evidence is given 
to prove that blood-serum has an inhibitory action on 
cell-division; and it will also be seen that it is pos- 
sible to measure this inhibitory action. Since the 
cell-proliferation of healing is caused by chemical 
substances contained in the soluble remains of dead 
tissues, and since, as will be shown, bacteria decom- 
pose these solutions, a field of research is opened for 
the investigation of this decomposing action by various 
pathogenic bacteria; for in decomposing the sources 
of the causes of healing they greatly modify that pro- 
cess, and the healing process must play an important 
part in immunity against disease. Further, bacteria 
may have an action on the substances contained in 
blood-serum which restrain cell-division. We fear that 
we have hitherto been able to do little towards the 
investigation of this factor in the problem of immunity, 
which is now mentioned for the first time. 

These are only a few of the fields which have been 
pried into by experimentation with this new method. 
It has been impossible for us to investigate all the 
paths of research which have been opened up, and 
prospective workers may be assured, from our own 
personal experience, that research with stained living 
cells will amply repay the time and patience expended 
on it. 

For the examination of the arrangements of the 
cells in living tissues we have not, so far, been able 
to make this in-vitro method so useful as is the older 
method of examining sections of dead tissues, but we 
think that improvements may be possible. For blood- 



10 THE SCOPE OF THE NEW METHOD 

examination, on the other hand, it takes one into a 
different realm compared with the older methods. 
Examined by the older methods, a cell appeared 
usually as a flattened, stained diagram; by the new 
one it appears as a sphere. The difference is com- 
parable to that which exists between an old Japanese 
print in which there is no perspective and a perfect photo- 
graph seen through a stereoscope. By the older meth- 
ods, for instance, the nucleus of a lymphocyte appears 
as a flattened, homogeneously stained mass, or perhaps 
the stained chromatin resembles a "spireme" within 
the nucleus; by the new method it is seen at a glance 
that the nucleus in the living cell is a round, trans- 
parent ball, studded on its outside by minute chromatin 
granules. There is no doubt that the observation of 
the living cell is a new study. In almost every slide 
one sees something of interest which has not been 
seen before. Living cells seem to have small points 
of individuality which can only be seen when they are 
stained alive. 

Take for example the phenomena of cell-division. 
The mitotic divisions, although the same in general 
principles (unless of course we take steps to induce 
asymmetrical divisions by an alkaloid) are almost 
always slightly different, depending to some extent 
upon the stage of division reached, and upon the 
attitude in which the cell happens to be presented to 
the observer. 

By this means of cytological study we may frankly 
say that we cannot tell what revelations may turn up 
at any time. This book will record a few of them, but 



APPLICATION TO CANCER RESEARCH 11 

there are doubtless many more to come. The feeling of 
astonishment may be imagined when one of us for the 
first time — and the cells have been discovered for more 
than a century — saw most of the polynuclear leucocytes 
in the specimen in the act of division. It was expected, 
it is true; but the way in which these cells divide was 
by no means expected. 

We have carefully searched the literature relating to 
our subject, without discovering points which have 
helped us. Most of the literature is devoted to de- 
scriptions of morphology which are not of much 
assistance in this kind of experimental work. There 
is no literature dealing with the effects of chemical 
substances on stained, individual, living human cells, 
and if a point is to be unravelled we have found it 
better to make experiments for its solution rather 
than to depend upon any literature dealing with the 
observation of dead cells. 

The new investigator will have to begin at the 
beginning, which is not far off, and he will have to 
do so with an open mind. 

The foregoing points indicate briefly the scope of 
this book descriptive of the new methods, and of the 
paths of research which have been opened by them. 
But we shall also describe in detail the main path 
which we have followed — namely, the adoption of the 
methods for the elucidation of the cause of cancer. 
It must be obvious that since we can now induce 
proliferation in human cells, and since the proliferation 
of certain human cells is the fundamental condition 
which characterises cancer (for that is what it is), we 



12 THE SCOPE OF THE NEW METHOD 

can, by investigating the chemical cause of prolifera- 
tion, throw considerable light on the cause of cancer. 
Cancer is essentially a growth caused by excessive 
cell-proliferation, and the new methods are the only 
ones which have given us the power to induce an 
individual cell to reproduce itself. 

As will be seen later, we can say more than this, 
for we can induce by certain specific chemical agents 
those remarkable asymmetrical mitotic divisions in 
human cells which are characteristic of many of the 
divisions which occur during malignant proliferation. 
The latter part of this book will therefore relate to 
Cancer Research. 

Before closing this chapter, two other points must 
be mentioned. The usual cytological phraseology has 
been found to be difficult to apply to many of the facts 
seen by the new methods. For instance, the word 
"nucleus " has a very vague meaning, and yet every one 
uses it. It arose, we believe, from the examination of 
cells with the lower powers of the microscope, which 
are commonly employed in the study of "pathological 
specimens." The nucleus of a cell, studied from this 
aspect, is merely a deeply stained body within the cell; 
but in reality the nucleus is composed of several dif- 
ferent parts, each of which has a separate function 
during cell-division. The body which appears as the 
nucleus in some cells has a very different function to 
that which appears as the nucleus in others. For 
instance, the body which appears as the nucleus of a 
lymphocyte under low magnification forms the spindle; 
whereas what are usually described as the nuclei of 



CYTOLOGICAL DEFINITIONS 13 

leucocytes are their centrosomes. The so-called nuclei 
of leucocytes ought, we think, in reality, always to be 
called the centrosomes, and the word "nucleus" deleted 
from their morphology. We have done our best to 
retain the usual cytological terms in the senses in which 
they are usually employed; but we must ask some 
indulgence when referring to those cells in which 
divisions have been seen for the first time, and in which 
these divisions differ very materially from those which 
occur in other types of cells. Again, we use the defini- 
tion "amoeboid" for the exaggerated movements 
exhibited by cells under the influence of alkaloids, 
but it must be understood that these movements 
differ from the blunt and sedate amoeboid move- 
ments which are commonly seen — that is to say, 
they are far more exaggerated and are absolutely 
characteristic. 

We think that, from the persistent examination of 
dead structures, cytology has been rather led away 
into a maze from which it will be difficult to extricate 
it; and it is possible that pathology may have to be 
modified in some of its points now that we know 
a great deal more regarding the causes of the prolifera- 
tion of cells. 

The last point to which attention must be directed 
is, that one ought to be careful how attempts are made 
to demonstrate new facts observed by this method to 
other people. If the specimen is actually under the 
microscope, and other people are present, then, of 
course, a few persons can see the new fact. But these 
living cells never last long, and many has the occasion 



14 THE SCOPE OF THE NEW METHOD 

been that a few persons have seen, say a beautiful 
mitotic figure, when suddenly a later arrival at the 
microscope says that he can see nothing, and on exam- 
ination it has been found that the figure has completely 
vanished owing to the onset of achromasia. If other 
people wish to see any experiment, two or three should 
await beside the microscope ; but they may have to wait 
a long time before a typical specimen is found, for, as 
has been pointed out, cells rarely present exactly the 
same appearances every time. It is of common occur- 
rence that on one day perfect specimens continually 
present themselves, but on the next every cell appears 
to be distorted, or always in the wrong position. For 
this reason we have found it better to take photomicro- 
graphs and convert them into lantern slides rather than 
attempt demonstrations to many people. 

It is right to mention that this method requires the 
expenditure of patience and time on the part of the 
investigator. One cannot attain good results in a few 
minutes, but if some time is devoted to it the value of 
this in-vitro method will be appreciated. 



CHAPTER II 

THE GENERAL PRINCIPLES OF THE METHOD THE 

APPARATUS REQUIRED THE SPECIAL PHOTOMICRO- 
GRAPHS APPARATUS THE REVOLVING APPARATUS. 

This method by which cells are observed in vitro is 
very simple. They are placed on a film of agar jelly, 
which holds in solution any material with which we 
may wish to experiment. To prepare the film, a drop 
of molten jelly is poured on to a slide, which is then 
laid on a level surface until the jelly sets firmly. A 
drop of the citrate solution in which, say, blood-cells 
are suspended is then placed upon a cover-glass, which 
is inverted and allowed to fall flat on the film. It 
might be thought that the weight of the cover-glass 
would be sufficient to kill the cells; but they sink into 
the jelly to some extent, and so become protected. 
Before this happens, however, they spread out centri- 
fugally from the centre to the periphery of the cover- 
glass, and if a drop of blood be examined in this way 
on stain-containing jelly they may be seen by the 
naked eye rushing in every direction towards the edges 
of the cover-glass. When this movement has ceased, 
if the slide is held up between the observer and the 

15 



16 THE GENERAL PRINCIPLES OF THE METHOD 

window it will be seen that the surface of the jelly 
over which the cells have passed is studded with 
corpuscles. 

If the jelly has been properly made the slide may 
be handled freely. It may be tilted to any angle, and 
even turned upside down without the cover-glass sliding 
off or the jelly becoming displaced. This is a fortunate 
fact, because it enables the microscope to be placed 
at any convenient angle for examination of the slide 
or for purposes of photography. If the specimen is 
quickly focused under the microscope while the 
spreading-out process of the cells is going on, using 
a J-inch objective and, say, a No. 4 eye-piece, the 
picture presented is a very remarkable one. The cells 
will be seen rushing along in a direction from the 
centre of the cover-glass towards its margin; they 
tumble over each other, leucocytes and red cells, 
lymphocytes and blood-platelets, bumping into each 
other and apparently all striving to reach some imagin- 
ary goal. Gradually the flow becomes slower and 
slower, the cells cease to "barge" into each other so 
fiercely, they squeeze past one another, and it will be 
realized what a marvellous power blood-corpuscles 
have of accommodating their shapes to almost any 
requirements. 

Leucocytes and red cells all behave in the same 
way. They allow themselves to be squeezed through 
gaps between other cells, which appear to be so small 
that if it had not actually been seen one never would 
believe it. As the flow becomes slower it will be 
seen that suddenly a passing leucocyte goes "ashore"; 



PREPARATION OF THE SPECIMEN 17 

its course is arrested because it has adhered to the 
jelly, or between the jelly and the cover-glass. Some- 
times the rest may be only momentary, when the cell 
may be seen to revolve on its own axis for a few mo- 
ments, and then pass on again in the slowing stream. 
Leucocyte after leucocyte afterwards becomes arrested 
in this way; they apparently stop first because they 
are larger and more "sticky." Then the red cells 
gradually stop, until at last the field is dotted with 
living blood-corpuscles, which may happen to become 
arranged in groups or rest singly side by side. 

The specimen may now be moved about by means 
of the mechanical stage, when it will be seen that all 
the cells in the film of blood under the cover-glass 
have become arranged in a manner very suitable for 
examination. The frontispiece of this book is a 
photomicrograph of a typical field presented by this 
method. 

The living cells all come to rest in a short time, and 
each one has its own share of jelly-surface, from which 
it has no alternative but to absorb any substances 
which have been previously dissolved in the jelly. 
Having focused a field, therefore, which contains an 
example of the cell with which one wishes to experi- 
ment, it is only necessary to wait until that cell has 
sufficiently absorbed the contents of the jelly for it to 
respond to the agent which has been dissolved in it. 

"Artefacts" do not exist; the surface of the jelly 
is the same all over. One has no control over the 
attitude which a cell may adopt, no matter what 
part of the jelly-surface it may come to rest upon, nor 



18 THE GENERAL PRINCIPLES OF THE METHOD 

over the other cells which form its immediate sur- 
roundings. The cells are always placed on the jelly 
in identically the same way as has just been described, 
and therefore the only way in which one can in- 
tentionally affect the individual cells is either by 
deliberately (1) mixing some other substance with the 
jelly before it is set on the slide, or (2) by keeping the 
slide at various temperatures. It sometimes happens 
that unintentionally the cells may become distorted 
by the presence in their neighbourhood of some for- 
eign substance which has been accidentally mixed with 
them in the citrate solution in which they have been 
suspended prior to being placed upon the film; but 
such a foreign body may easily be recognized. 

The apparatus required for these researches is not 
very elaborate. Many of the earlier experiments were 
made in a cabin in a battleship, where there is not 
much room for scientific apparatus, but we simply 
enumerate them here for the benefit of those who may 
desire to commence the study of in-vitro methods for 
the first time. They consist of: 

1. Microscope slides. 

2. Cover-glasses. These should be very thin and 
| of an inch in diameter. A few larger ones, say § of 
an inch, may occasionally be needed. A silk hand- 
kerchief is required to polish the cover-glasses, which 
should be very clean and kept in alcohol. 

3. Capillary glass tubes. These are constantly in 
use, and it is well to begin with a stock of 100 of them. 
They should be about 4 inches long, having an internal 



GENERAL APPARATUS 19 

diameter of 2 millimetres, and should be kept in water 
which has been sterilised. 

4. A watch-maker's file for removing the sealed 
ends of the capillary tubes. 

5. Hair-lip pins are most convenient for pricking 
the finger or the ear to obtain the blood. 

6. Two or three needles in handles for teasing out 
tissues, etc. 

7. Pipettes; several 1-cc. pipettes, graduated in lOths 
and lOOths; a graduated 10-cc. pipette, and one or 
two ungraduated of 5-cc, 3-cc. and 2-cc. -capacity. 

8. Two beakers. These are used for boiling water 
in. The jellies are melted and made liquid by im- 
mersing the test-tubes containing them in water which 
is boiling in the beakers. 

9. Tripod stand and gauze cover. 

10. A Bunsen burner or good spirit-lamp. 

11. A 100-ec. graduated measure. 

12. Two small flasks. 

13. Some glass funnels and filter paper. 

14. A selection of test-tubes. 

15. A centrifuge. 

16. An ordinary chemical Centigrade thermometer 
for recording the room temperature. 

17. A good incubator, which should maintain a 
temperature of 37° Centigrade, i.e. the temperature of 
the blood. Hearson's is a very good one, but any of 
the ordinary water-jacketed types will do. An auto- 
matic thermostat is a convenience. 

18. The microscope is the most important part of 
the outfit, and it should be a good one. 



20 THE GENERAL PRINCIPLES OF THE METHOD 

This work consists largely of cytology, requiring 
accurate observation as to details, and the highest 
powers of magnification. Any good microscope stand 
will do, but we think that the English tripod one 
is the best, especially if the special photomicrographic 
apparatus is adopted, in which case it is almost es- 
sential. The larger and heavier the stand the better. 
It must have a mechanical stage, which should be built 
with the instrument; not an "attachable" one. The 
lenses must give good definition. Two objectives 
only are necessary — a sixth-inch, and an immersion 
twelfth. We use equivalents of these in a Zeiss D, 
and a Zeiss 2-mm. apochromatic lens, which is com- 
pensated for the long-draw-tube of 250 mm., and which 
has a numerical aperture of 1 . 30. There is no doubt 
that an apochromatic objective for this work is vastly 
superior to an ordinary twelfth-inch lens, especially if 
photography is to be used. 

The eye-pieces we employ are the No. 4 and No. 8 
Zeiss compensated ones, and these, or their equiva- 
lents, will be found most useful. 

The light should always be artificial; daylight is 
not suitable for this method. We have found that the 
inverted incandescent gas-burner gives the best light 
for ordinary work, or if electricity is preferred, the 
1 -ampere Nernst lamp is most suitable. If neither 
gas nor electricity are available, the spirit-lamps which 
give a light by heating an inverted mantle have proved 
most suitable in our hands. No matter which light is 
used, it is better always to use the same, in order that 
contrasts may be detected readily. 



SPECIAL APPARATUS '21 

It is well to remember that with this method one 
cannot afford to waste much time in manipulating the 
adjustments of the microscope. The cells, under some 
conditions, die quickly, and we therefore have to search 
the specimen very rapidly before "achromasia" occurs, 
when all the cells vanish, as will be presently described. 
It is better, therefore, to have everything ready before 
the specimen is prepared. 

The microscope should be fitted with a nose-piece. 
so that the objective can be changed quickly. When 
using the immersion lens, great care must be exercised 
in placing the drop of cedar oil on to the cover-glass, 
for the cells and jelly-films are easily destroyed if it is 
accidentally touched with the solid oiler. There is 
neither time nor necessity to reverse the mirror from 
concave to plane when the objective is being chai _ 
from a dry to an immersion one. When searching 
through the specimen- of living cells, rapidit; 
focusing will be found to be of more value than too 
much attention to accurate microscopy, which is 
difficult, if not impossible, to adhere to with this 
method. The focusing of the substage condenser on 
the specimen cannot be very accurate. Most micro- 
scopes are adjusted for slides of a certain thickness. 
but we have to place a comparatively thick film of 
jelly on top of the slide, and hence the objective 
is always farther away from the condenser than it 
ought to be. 

The photomicrographic apparatus (figs. 2-5 in- 
vented for this method has been designed so that a 
photograph can be obtained quickly of any field in 



22 THE GENERAL PRINCIPLES OF THE METHOD 

a specimen without disturbing either the microscope 
or the specimen. Having obtained the negative, the 
camera is removed in a moment and the examination 
of the particular cell or specimen under observation 
can be immediately proceeded with in the usual way. 
The old forms of cameras which necessitated the moving 
of the microscope or the specimen are not useful for 
recording specimens of living cells. An instrument 
is required capable of being immediately connected 
with the microscope as it stands, so that two or three 
records of the same cell may be taken before it dies or 
becomes achromatic and vanishes. It is necessary to 
use a powerful light, and the light itself will kill the 
cells if they are exposed to it for very long. For this 
reason we employ a powerful light for the photography 
and another for the eye Avork, but each of them fixed 
and capable of being used independently of one another. 
The inverted gas-burner above referred to, being placed 
at a distance of two feet above the mirror, gives a soft, 
indirect illuminant for ordinary work, the other being 
a powerful electric Nernst burner, which is placed 
behind (that is, underneath) the mirror. When a 
photograph is to be taken the mirror is swung aside, 
and the light from the Nernst lamp replaces that from 
the gas one. 

The microscope is fixed on the bench and tilted 
at an angle of about 45° from the vertical. All 
the microscopes which we use are bolted perma- 
nently on to the bench, and they can only be 
moved with the aid of a screwdriver. The instru- 
ments are not placed vertically, but are tilted at 



SPECIAL APPARATUS 



23 




03 — 3 

— & 

C fl r. 

1l.al 



■-jsg. 



r — — > 

O? DC Pi 



G) L 



o 

51. 



SPECIAL APPARATUS 



25 




— 




- 




OH 






<D 












"r. 


CD 

— 


U 


, o 


g 


CO 




O 


£ 


Sh 

o 


02 




CO 


i 








a 


o 


— 




O 


'g 












*j 


-o 




a> 




+3 








a; 




CO 


>; 


_C 








co 


c 




- 


eS 






bjc a; 





s 


o 


C3 




O 


p 


a> 




















O 


>. 


-u 




73 


X 


<D 












~Z 




a 


co 


-^> 


3 


■*^ 


-^> 


a 


c3 


CD 


tf 


o 


— 


<D 


c_ 


ft 






* w 


<D 


03 


>. 


— 


CD 



SPECIAL APPARATUS 27 

an angle, because this is most convenient for comfortable 
use. This last point is most important, for one may 
have to spend hours searching through films with this 
method, and it is most wearying to have to work in an 
uncomfortable position. 

Behind the mirror, and standing a little way back 
from it, there is a Nelson's aplanatic condenser (Wat- 
son) with iris diaphragm, and immediately behind 
this again is fixed a rectangular all-glass water-tank. 
This small tank has an outlet pipe above, and an inlet 
pipe below, connected by means of rubber tubes with 
a sink and a cold-water supply respectively. The water 
is kept circulating through this tank when the apparatus 
is in use. Lastly, behind the tank is the burner of 
a 1 -ampere Nernst lamp. 

Above the microscope and set at an angle corre- 
sponding to its tilt a rigid wooden board is arranged, 
being fixed to the ceiling above and, by means of a pair 
of legs on either side of the microscope to the bench 
below. The board, which is about ten inches in width, 
by seven-eighths of an inch thick, has a slot cut into it 
in which a box camera can easily slide up and down and 
be capable of being fixed at any point by means of a 
screw clamp. The camera is fitted with a shutter 
(instantaneous and time exposures) the aperture of 
which is connected with a "high-power projection eye- 
piece" (Watson) by means of a flexible velvet collar. 

The Nernst burner, the cooling tank, the two 
condensers, and lastly the camera must all be very 
carefully centred to the microscope, and immovably 
fixed so that the whole apparatus may always be ready 



28 THE GENERAL PRINCIPLES OF THE METHOD 

for use, the Nernst lamp being kept lighted as well as 
the gas one during any experimentation in order that 
a photograph can be taken at a moment's notice. Of 
course, so long as the mirror is in its usual position no 
light reaches the specimen from the Nernst lamp; 
swing the mirror out of its position, and the light is 
instantly changed from that of the gas-burner to the 
powerful one from the Nernst burner. The distances 
between the Nernst lamp, aplanatic condenser, and the 
substage condenser, are of great importance. It must 
be determined at the outset by trials which distances 
give the best results. The presence of the water-tank 
renders it difficult to make a rule. 

When a cell or other object comes under observa- 
tion which it is desirable to photograph, the working 
eye-piece is removed from the microscope draw-tube; 
the camera is allowed to slide down the beam until its 
shutter is about an inch from the mouth of the draw- 
tube, when it is clamped to fix its position. The 
projection eye-piece, which is already attached to the 
camera-shutter by means of the flexible velvet collar, 
is inserted into the microscope draw-tube. The mirror 
is now swung on its gimbals out of the focal axis, 
thus allowing the light from the 300-candle-power 
Nernst burner to replace that of the gas-burner; and 
the former, after being cooled by transmission through 
the intervening water-trough, is projected directly 
through the two condensers. The image of the field 
of the specimen will then be seen on the ground- 
glass screen at the back of the camera, where it can 
be rapidly focused. 



SPECIAL PHOTOMICROGRAPHY 



29 




Fig. 4. — The photomicrographic 
water-cooling tank and Nernst burner, 
for direct observation. 



apparatus. Showing positions of 
The microscope mirror is in position 



SPECIAL PHOTOMICROGRAPHY 



31 




Fig. o. — The photomicrographic apparatus. The microscope mirror is 
swung aside for photography. 



SPECIAL PHOTOMICROGRAPHY 33 

If preferred, focusing may be done with a lens; 
but in the case of a specimen of blood, the edges of 
the red cells afford a good indication of its accuracy, for 
they seem just to disappear when the accurate focus 
is obtained. When they are out of focus the edges of 
the cells stand out in high relief. Having obtained the 
focus — and stress must be laid on this point — the cell 
or other object is deliberately thrown out of focus to the 
extent of about 6+0 th of a millimetre 1 by screwing down 
the fine adjustment so as to bring the objective nearer 
the object. The reason for this is that the cells are 
resting on a jelly under a cover-glass which is all the 
time slowly sinking into the jelly, and, of course, 
carrying the cells with it. The latter, therefore, are 
sinking out of focus all the time. By deliberately 
"over-focusing," when the exposure is actually made 
the focus will become accurate, and the sinking of 
the cover-glass compensated for. 

The length of the exposure varies with the objective 
used and the candle-power of the light, which in its 
turn varies with the voltage. It is best to find the 
length of the exposure by experiment, but we give 
about twenty seconds with the apochromatic objective, 
using "backed" Imperial plates. The water cooling 
tank cuts out light, but it is very necessary to use it 
in order to delay death of the cells and the onset of 
achromasia, both of which are accelerated by heat 
rays. The tank cuts off some of the heat rays, but 
allows the passage of the actinic ones. Many specimens 

1 The fine adjustments of most microscopes are graduated to allow of this 
measurement. 

3 



34 THE GENERAL PRINCIPLES OF THE METHOD 

were lost owing to achromasia before the cooling tank 
was employed. 

The photograph having thus been quickly taken, 
the mirror may again be swung into position, the 
camera pushed out of the way, and, having inserted 
the working eye-piece, the examination of the specimen 
may be proceeded with, or other fields explored. We 
have taken a negative in fifty seconds with this appa- 
ratus, and as many as five negatives have been taken 
from different fields in a single specimen; but such 
speed is not often necessary. 

All the photographs which illustrate this book have 
been taken with the apparatus just described. It never 
gives trouble, and has proved most useful in supplying 
a means of recording the "specimens." It used to be 
most annoying to see unique mitotic figures or other 
interesting specimens slowly vanish before one's eyes 
without being able to record them satisfactorily. In 
fact, the best mitotic figure I have ever seen in a 
lymphocyte was induced before we possessed a camera; 
and although thousands of figures have been seen 
since then, I have never seen a picture comparable 
to it. It was seen by Professor Harvey Gibson as 
well as by myself. 

There is one other useful piece of apparatus which 
requires mentioning, viz. the "revolving apparatus." 
This is a simple clock-work contrivance which keeps 
a long test-tube revolving on its long axis. The 
test-tube is placed horizontally. The object of the 
appliance is to keep the blood-cells constantly moving 
in the "citrate solution," or other medium in which 



THE REVOLVING APPARATUS 35 

they may be suspended, while samples are under 
examination. If the capillary tubes containing the 
specimen are laid for some time on the table, the 
corpuscles will sink to the most dependent part of 
the citrate solution, and will ultimately adhere to the 
glass. By placing the tubes in the "revolving appa- 
ratus" this is effectively prevented. It is a good thing 
to have in the laboratory, for it delays loss of vitality 
in the cells; but it is not essential. 



CHAPTER III 

THE PREPARATION OF THE JELLY FILM 

Agar, the substance used for making the film on 
which the cells are examined, is obtained from sea- 
weed. It is very cheap, and may be bought in strips 
or as a powder. We have used Merck's powdered 
agar, which is quite neutral and pure. It is insoluble 
in cold water, but immediately soluble in boiling water. 
This solution, therefore, on cooling, sets as a jelly. 
It is necessary to have a stock of jelly constantly in 
hand, and a 2-per-cent preparation is used throughout. 
This will melt when its boiling-point is approached, 
but will not set again until the temperature has fallen 
almost to 40° C. Unlike gelatine, this jelly may be 
boiled over and over again, and it will always set at 
its usual temperature. 

The jelly is made in 2-per-cent strength for the 
reason that it will stand diluting with its own volume 
of water or other solution, and will still set as a 
jelly — that is, a 1-per-cent solution of agar jelly will 
set on a slide in the form of a film as it cools. By 
using the 2-per-cent preparation we are enabled 
to add an equal volume of any solution we please, 

36 



SALTS ARE NECESSARY 37 

so that the result is that the 1-per-cent jelly may 
contain quite a variety of substances, and if some 
human cells are placed on its surface we may try 
the effect on those cells of any of those materials 
which have been added in solution to the 2-per-cent 
agar. We are thus able to investigate, by a method 
which is simplicity itself, the effects of drugs or 
chemical substances upon the individual human cell. 

Before we begin to discuss this subject, however, 
we must be certain that the cells are alive when they 
are being subjected to the drug. It is, of course, 
well known that when, say, a drop of blood is re- 
moved from the finger the leucocytes are alive; but 
it is necessary to be certain that they are not killed 
immediately they are placed on the jelly-film. As 
will be discussed at greater length later on, we can 
always ascertain whether white blood-cells are alive 
or not by mixing a certain quantity of an alkaloid 
with the jelly; for alkaloids excite amoeboid move- 
ments, and it is obvious that these movements cannot 
occur in a dead cell. Since alkaloids have supplied 
the means of determining this point, we have also 
been able to ascertain how to make the jelly so that 
it will keep the cells alive as long as possible; for it 
is clear that a jelly which will allow cells to remain 
excited for the longest period with a given quantity of 
alkaloid must be the best jelly for keeping the cells 
alive when made without the alkaloid. The presence 
of a combination of certain salts is essential. 

Suppose a drop of blood is placed on to a film 
of jelly which contains only agar and water and no 



38 THE PREPARATION OF THE JELLY FILM 

salts. The red cells will hsemolyse immediately. The 
white cells are worth watching. As soon as they 
come to rest, or even before that, the polynuclear 
leucocytes seem to swell up, the granules exhibit 
"furious" Brownian movements, and in a few moments 
the cell totters and then bursts. Water kills blood- 
cells instantly if there are no salts present. Let the 
experiment be repeated, but, instead of using merely 
agar and water, now make the jelly with sodium 
chloride in the strength of "normal saline solution." 
It can be made thus: Melt a few cubic centimetres 
of 2-per-cent agar jelly and place 1 cc. in a test- 
tube. Prepare a solution of 1 . 8-per-cent sodium 
chloride in water. To the 1 cc. of molten 2-per- 
cent agar jelly add 1 cc. of the sodium-chloride 
solution. The test-tube will now hold 2 cc. of a 
1-per-cent agar jelly containing 0.9-per-cent sodium 
chloride, i.e. "normal saline solution." The whole 
is melted again, and a drop poured on a slide. If 
some blood is now examined on this jelly, it will be 
seen that the red cells do not "lake" immediately. 
The leucocytes, however, again die very quickly, as is 
seen by their swelling up, the onset of "dancing" 
movements of the granules, and by rapid bursting, 
although the rupture will not be quite so rapid as 
when only water was present. 

Now let the experiment be repeated a third time, 
but instead of adding a solution which contains only 
sodium chloride, let it contain in addition some sodium 
citrate, thus: To 1 cc. of 2-per-cent agar jelly add 
1 cc. of a solution containing 1 . 8-per-cent sodium 



STOCK SOLUTION OF AGAR 39 

chloride, and 2-per-cent sodium citrate. 1 (When this 
jelly is spread on the slide it will contain 1-per-cent 
agar, 0.9-per-cent sodium chloride, and 1-per-cent 
sodium citrate.) The picture presented by blood spread 
on such a film is very different from those in the last 
two experiments. The red cells are not crenated, but 
are beautifully spread out. The leucocytes are not 
dead, but alive and amoeboid; no Brownian movements 
of the granules can be seen, and the cells do not burst; 
on the contrary, they will live now for an hour or more. 
It may therefore be said that for the examination of 
living blood-cells (and it has been found that it is also 
the case for all cells yet tried) the jelly must always 
contain a certain amount of the salts sodium citrate 
and sodium chloride. "Normal saline" is not enough 
by itself. Cells die immediately when they are resting 
on a surface which contains only sodium chloride. 

These three experiments will prove instructive for 
the beginner with this jelly method, for they demon- 
strate how the jelly is prepared. It must be observed 
that for the purposes of these researches the supply 
of jelly is always kept as a 2-per-cent solution of agar. 
When, however, it is placed as a film on the slide, it is 
always diluted with an equal volume of some other 
solution, so that the film invariably contains 1 per cent 
only of agar. It is in the diluting solution (always 
added in an equal volume) that the salts, and any other 
substances to be experimented with, are contained, and, 
obviously, before being added to the agar they must 
be of twice the required strength so as to be reduced 

1 Potassium oxalate may be substituted for sodium citrate. 



40 THE PREPARATION OF THE JELLY FILM 

to the proper one in the resultant jelly with which the 
film is made. It is imperative to explain the way in 
which the jelly is made even at the risk of being 
verbiose. Bear in mind, therefore, that two solutions are 
required — namely, No. 1, a stock 2-per-cent solution 
of agar, and No. 2, a solution which contains the 
other substances the effects of which are to be tried on 
the cells. Solutions Nos. 1 and 2 are always mixed 
together in equal parts and then boiled up to form 
No. 3, from which the jelly-film on the slide is prepared. 
No. 1 is always the same. No. 2 may contain a variety 
of substances, but no matter how much of any sub- 
stance No. 2 may contain, No. 3 will always have half 
that amount. For example, if one wishes a cell to rest 
on a jelly containing 1 per cent of morphine one must 
have 2 per cent of morphine in No. 2, so that when the 
two solutions are mixed in equal parts the combination, 
that is No. 3, will contain 1 per cent of morphine. 

A word is necessary as to the effects on cells of the 
agar itself. It appears to be innocuous. We have tried 
it in strengths double and even four times as great as 
that contained in the stock solution, without apparently 
producing any deleterious effect upon the vitality of 
the cells experimented with. 



CHAPTER IV 

CELLULAR STAINING, DEATH, AND ACHROMASIA 

By far the larger number of cells examined in these 
researches have been blood-cells taken from the finger. 
The white blood-corpuscles have offered a very interest- 
ing study, and since they respond to chemical agents in 
a way very similar to those observed in several other 
varieties of cells, and, since they are very easily obtained 
and can be very carefully watched, it is convenient to 
describe what we have seen with them. These cells 
play an important role in the phenomenon of healing, 
and ultimately go to form some of the fixed tissue- 
cells, especially after an injury has been sustained. 

For the examination of blood-cells in in vitro it is 
best first to mix the sample of blood (which should be 
drawn freshly from the finger) with an equal volume of 
"citrate solution." 1 The citration of the blood not 
only prevents it coagulating, but it also keeps the cells 
alive sometimes for as long as seven days. 

The way in which we citrate the blood is as follows: 
One end of a capillary tube, such as has been described 
(Chapter II.), is dipped into the citrate solution, some of 

1 Three-per-cent sodium citrate, and 1-per-cent sodium chloride. 

41 



42 CELLULAR STAINING, DEATH, ACHROMASIA 

which runs up into it. The amount drawn into the 
tube can, if necessary, be controlled by keeping the 
finger on the other end. It has been found most service- 
able to allow the solution to fill the tube to the extent 
of about half an inch, and any excess can always be 
removed by tapping the lower end of the tube upon the 
table, which causes some of it to run out. Having got 
a sufficient quantity of citrated solution into the tube, it 
is run down to one end of it, and a mark is made at the 
upper limit (or meniscus) with a grease pencil. The 
fluid is now run along the tube by depressing its other 
end until its lower meniscus stands at a level of the 
mark, and a second mark is then made at the upper 
meniscus, after which the tube is again placed vertically 
so that its contents runs down to its original position. 
The finger having been pricked, a drop of blood is 
squeezed out and at once allowed to run into and mix 
with the citrated solution in the tube, the greatest care 
being taken that no air-bubble intervenes between the 
fluids. The blood should be allowed to run in until 
the upper meniscus of the mixed fluids reaches the 
upper mark. Thorough mixing of the blood with the 
citrated solution is ensured by rocking the tube in such 
a way that its contents runs from end to end. The 
mixture in the capillary tube will now consist of equal 
proportions of blood and citrate solution, and of this a 
drop is tapped out on to a cover-glass, which is then in- 
verted and allowed to fall on the agar film in the usual 
way. When tissues are to be examined, a small portion 
of the growth or normal structure is either teased 
out or scraped into a little of the citrate solution in 



THE STAINING OF A CELL 43 

a watch-glass. A drop of the cell-containing mixture 
is then placed on to the cover-glass and similarly placed 
upon the jelly. 

The citrate solution simply acts as the vehicle 
in which the cells are kept in a living condition before 
being placed upon the jelly, and furthermore, by dilut- 
ing the blood, it reduces the actual number of cells 
which come to rest in any field of the film. If no 
citrate solution is used they are apt to become huddled 
or crowded together owing to their great numbers, 
and the leucocytes may become completely hemmed 
in by erythrocytes so that a clear observation of the 
whole cell cannot be obtained. 

We must now pass on to the study of some of 
the phenomena connected with the staining of the 
cells, which have been the means of elucidating many 
cytological details which have led to the correct 
appreciation of the effects of chemical substances on 
cells. In this chapter, however, I do not propose to 
discuss very deeply the actual laws by which the 
staining of the cells is controlled; that will be reserved 
for discussion when I come to speak of the diffusion 
of the substances, including stain, into the cells. In 
the meantime I shall simply describe what happens 
to the cell as it absorbs the stain (say Unna's 
polychrome methylene blue, Grubler) ; how the stain 
causes the gradual death of the cell (the staining of 
the nucleus invariably kills it) and how death is 
followed by achromasia. The amount of stain which 
is put into any given jelly is not added in a hap- 
hazard way, the actual amount necessary to cause 



44 CELLULAR STAINING, DEATH, ACHROMASIA 

leucocytes to stain deeply in a given time being 
a very definite one, as will be described in the next 
chapter; but in the meantime we must assume that 
a jelly has been correctly prepared containing, besides 
the proper proportions of sodium citrate and sodium 
chloride to keep the cells alive, the proportion of stain 
requisite to enable us to observe its gradual passage 
into the leucocytes as they absorb it. 

The agar jelly, of course, will be coloured purple 
owing to the stain it holds in solution, but it will be 
quite transparent and will allow sufficient light to 
penetrate it so that the cells may be clearly observed. 

Having without delay placed the film, with the blood- 
cells upon it, under the microscope, at first the cells 
will be quite unstained, but the white corpuscles 
may easily be recognized owing to their granulation 
and size. Let a polymorphonuclear leucocyte be 
watched. Gradually its granules become tinted a 
faint red colour (fig. 6) and about the same time 
amoeboid movements may begin. If certain propor- 
tions of alkaloid have been added to the jelly, these 
amoeboid movements will be very marked (fig. 7). 
The staining of the granules becomes deeper and 
deeper, always maintaining the same bright scarlet 
colour. In spite of the deepening coloration of the 
granules, amoeboid movements will continue, showing 
that the cells are alive and that their vitality is appar- 
ently unaffected by the staining of their granules. 
It is not only the polynuclear leucocytes that behave 
in this way, but the mononuclear, or lymphocyte, 
cells as well. 



A STAINED NUCLEUS MEANS DEATH 4-5 




Fig. 6. — The granules of the leucocyte are gradually becoming stained. The 
red cells are unstained. Low power. 





Fig. 7. — The leucocyte's granules are stained. Its nucleus is unstained. 
The pseudopodia are extruded in response to atropine, which is diffusing 
into the cell as well as the stain. 



A STAINED NUCLEUS MEANS DEATH 



47 




Fig. 8. — The same field as 7. The leucocyte is retracting its pseudopodi 

■ 




Fig. 9. — The same field as 7 and 8. The retraction of pseudopodia is 
nearly complete. The lobes of the nucleus of the leucocyte are turning a 
faint blue colour. 



A STAINED NUCLEUS MEANS DEATH 49 




Fig. 10. — A leucocyte excited by atropine. Its granules are deeply stained, 
and its nucleus is also beginning to stain a blue colour. Low power. 









Fig. 11. — A leucocyte which has just been killed by the staining of its 
nucleus. Its granules are also deeply stained. 



A STAINED NUCLEUS MEANS DEATH 51 

After a short time the extrusion of pseudopodia 
ceases, and it will then be noted that general retraction 
(figs. 8, 9) of pseudopodia begins to occur. In the 
meantime the lobes of the nuclei of the polynuclear 
cells begin to turn a faint blue colour (fig. 10). If 
two or more leucocytes happen to be in the same 
field, it will be seen that they all behave in a like 
manner, for the stain affects them all equally. In a 
few moments all the amoeboid movements cease, for 
death is about to occur, and then, sometimes quite 
suddenly, the nuclei turn bright scarlet (fig. 11) and 
the death of the cell takes place. 

We have never yet seen a cell show any amoeboid 
movements when its nucleus has stained scarlet. By 
mixing some blood with a citrated solution of stain 
one can cause first the granules and then the nuclei 
of the leucocytes to stain, the difference depending 
on the length of time the mixture has been made. 
If now cells with only their granules stained are placed 
on to a jelly-film which contains an alkaloid, we can 
excite these cells, showing that they are alive. But 
if their nuclei are stained scarlet, no excitation or move- 
ments of any sort can be produced, and there can be 
little doubt, therefore, that the staining of the nu- 
cleus kills the cells. White blood-corpuscles do not 
seem to mind the staining of their granules; but the 
staining of their nuclei invariably causes their death. 
This is a rule to which we have never yet seen an 
exception in any cell which we have examined. A 
stained nucleus is incompatible with life. 1 

1 We have tried several stains, but this rule holds good with them all. 



52 CELLULAR STAINING, DEATH, ACHROMASIA 

When the lobes of the nuclei stain scarlet, the 
chromatin network within them shows up well. The 
blue coloration which precedes the scarlet one is due, 
I think, to the staining of the nuclear wall. The 
polychrome dye contains two stains, a red and a blue 
one, and the nuclear wall seems to have an affinity 
for the blue one, while the chromatin combines with 
the red. The staining of the nucleus, therefore, is a 
sign that the cell has died, and one now sees a circular 
dead cell (in reality it is a spherical cell which has 
become flattened out) with its granules stained scarlet, 
and in their midst there is the polylobed nucleus, also 
stained scarlet. Let the specimen be watched still 
further. Gradually the cell-wall is seen to bulge out 
in places (fig. 12), apparently away from the granules. 
After a few moments this bulging becomes general 
(fig. 13), and the cell presents a clear halo of cell-wall 
and cytoplasm outside the limit of the mass of granules 
in its centre. This is due to the gradual liquefaction of 
the cytoplasm which occurs at death, beginning at the 
periphery and progressing slowly towards the nucleus. 
Sometimes a few stained granules appear to migrate by 
the "dancing" Brownian movement into the liquid 
cytoplasm which has bulged out the cell- wall. Under 
suitable conditions the Brownian movement becomes 
general, showing that all the cytoplasm has liquefied — 
a certain sign of death. 

No matter whether the cytoplasm has completely 
liquefied or not, however, one of two things is bound 
to happen after a short time. The granules and nucleus 
may remain stained for half an hour or so, especially 



ACHROMASIA 








\ 



Fig. 12. — The leucocyte lias just died owing to the staining of its nucleus. 
The cell-wall is beginning to bulge because the cytoplasm is liquefying. 



V: 



Fig. 13. — The onset of achromasia. The same field as 12. The stain 
is beginning to fade from the nucleus. The bulging of the cell-wall has 
become general. 



AC H ROM ASIA 55 

if a temperature of about 30° C. is maintained, which 
may prevent the bulging of the cell-wall ; but after 
that time the cell will either burst and become 
achromatic, or become achromatic without bursting 
(fig. 14). In either case achromasia, or loss of stain 
from the cell, invariably occurs. If the temperature 
is low (say that of the room) the cell will probably 
burst and its granules will be scattered about on the 
surface of the jelly. Now, when a cell bursts on a jelly 
which contains salts — such as the one with which we 
are supposed to be experimenting — there is another 
rule to which there is no exception, namely, that the 
cell's nucleus loses its stain instantly. In a flash all 
coloration has gone from it. But the granules may 
remain stained for half an hour or more; and then 
they also gradually lose their stain (fig. 15), and appear 
slowly to vanish from the scene. The phenomenon 
of achromasia always overtakes the cells sooner or 
later. 

If the cell does not burst, the stain disappears, but 
its disappearance is much slower. This is a pretty 
phenomenon to watch; but it requires a warm room or 
warm stage. Suppose we are watching a cell which 
is dead, having its nucleus and granules stained bright 
scarlet. The stain gets a deeper colour, and one 
wonders how deep a shade it will attain to. Suddenly 
the staining seems to stop, and the depth of colour may 
remain the same for a quarter of an hour or so. Then, 
almost imperceptibly at first, the colour becomes paler, 
and with an accelerating speed the colour fades away 
from the lobes of the nucleus, until that structure 



56 CELLULAR STAINING, DEATH, ACHROMASIA 

remains as unstained as it was when the cell first came 
to rest on the jelly-film. After a few minutes the 
granules slowly lose their stain also, until nothing seems 
to remain. Ultimately the red cells disappear too (figs. 
14, 15), and the field, which a short time previously was 
dotted with red cells and stained leucocytes, now 
becomes a blank, and a new-comer looking at the 
specimen would hardly believe that there had ever 
been any cells under view. The picture afforded by 
the successive occurrence of the staining, death, and 
onset of achromasia in the cells is well worth seeing. 
First the slow diffusion of the stain into the cells, 
staining first their granules and then their nuclei; 
the gradual retraction of pseudopodia as the nuclei 
stain, and then the bright scarlet coloration of the 
nucleus itself as death occurs. After a pause the 
gradual fading of the stain, first from the nucleus and 
then from the granules, until at last nothing remains 
visible of the leucocyte in the place it filled among 
the neighbouring red cells. The whole phenomenon 
reminds one of a lantern dissolving view — the onset 
of staining, its climax, and then its disappearance. 
Achromasia invariably occurs after a time — nothing 
which we know of will prevent it; but heat greatly 
accelerates its onset, and a ruptured cell always becomes 
achromatic before a whole one. 

I do not propose here to give the details of ex- 
periments which I made some years ago, to try to 
investigate the nature of this phenomenon of achro- 
masia; they will be found in a paper, ''On the Cause 
of Achromasia," in The Lancet of Januarv 23, 1909. 



CAUSE OF ACHROMASIA Di 




\ 



Fig. 14. — Achromasia. The same field as 13. The stain has gone from 
the nucleus, although the granules are still stained. Note that the red cell 
is disappearing. 






Fig. 15. — Achromasia. The same field as 14. Many of the cell-granules 
have lost their stain. The cell-wall is nearly invisible. The red cell has 
disappeared. 



CAUSE OF ACHROMASIA 59 

I shall now simply state the conclusions which were 
arrived at then, subsequent experimentation not having 
altered my opinion in any way. 

Achromasia seems to be part of the general dis- 
organization which occurs in a cell after death. I have 
never seen the phenomenon in a living cell, and one 
cannot excite an achromatic leucocyte or lymphocyte. 
It is by no means necessary for a cell to be stained 
before it can become achromatic; on the contrary, one 
frequently sees dead cells which refuse to stain, although 
their living neighbours will stain well under suitable 
conditions. The rapidity of onset of achromasia de- 
pends upon the temperature and the presence and 
amount of salts. It also appears to depend to some 
extent on the completion of the liquefaction of the 
cytoplasm. The more advanced the liquefaction, which, 
of course, only occurs after death, 1 the more readily 
does achromasia take place. Heat and salts accelerate 
it greatly. If there are no salts present, even the 
nuclei of ruptured cells do not become achromatic for a 
long time. These stained nuclei may sometimes be 
seen floating about free from cytoplasm, granules, or 
cell- wall. I believe that achromasia is due to the 
chromatin passing out of the dead and liquefied cell by 
osmosis. If the chromatin is stained, the stain will dis- 
appear with the chromatin; if the cell is unstained, it is, 
of course, impossible to stain its chromatin if the latter 
has already passed out by osmosis. I believe that this 
is what has happened in the cell which is commonly 

1 See paper in Journal of Physiology, "On the Death of Leucocytes/' 
vol. 37, No. 4, 1908. 



60 CELLULAR STAINING, DEATH, ACHROMASIA 

known as the hyaline cell. Achromatic lympho- 
cytes resemble them strongly. A dead lymphocyte, 
from which the granules have disappeared, will not 
stain, and the cell resting on the jelly looks like a 
phantom. We have never been able to excite such 
a cell. If a specimen of fresh blood is placed carefully 
on a jelly-film, one does not usually see any such cells, 
for all the cells will stain; but after a while the film 
may contain many examples of achromatic cells which 
appear to be exactly like what are known as hyaline 
leucocytes by the older methods. Achromasia is a 
certain sign of death, and the recognition of its very 
characteristic appearances is of the utmost importance 
in this form of research. It should be borne in mind 
that a cell with a stained nucleus is dead, and so is 
a cell which is achromatic. 






CHAPTER V 

THE DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

THE "COEFFICIENT OF DIFFUSION" 

The following points regarding the diffusion of sub- 
stances into cells have been determined by experimen- 
tation with this method. A cell does not respond to the 
action of a chemical substance unless the substance has 
diffused into it. It is of the utmost importance, there- 
fore, that the laws which are concerned in this diffusion 
should be understood, both for the practical application 
of this in- vitro method in the study of the actions of 
substances on individual cells and also, I think, because 
it throws light upon the way in which drugs produce 
their effects upon the various systems and organs of the 
animal body. Very little has hitherto been known 
concerning the diffusion of substances into individual 
living cells; and although we do not claim to have 
advanced the knowledge of the subject to a very great 
extent as far as its scientific basis is concerned, we can 
safely say that we are now in a position to cause sub- 
stances to diffuse into individual cells according to our 
will. We do not know all the scientific facts, it is true, 
for more work will be necessary before these can be 

61 



62 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

determined, but we do know the more important laws 
which are sufficient for practical purposes. 

The diffusion of substances into living cells is purely 
a physical process. A cell does not seem able to exert 
any vital control or power of selection whatever over 
the diffusion of substances into its cytoplasm. In every 
cell with which we have experimented this has been the 
case, and if proof is needed it is afforded by the fact 
that we can at will cause cells to be excited, to repro- 
duce themselves, or to die, by employing the knowledge 
of the laws, which shall presently be described, enabling 
us to make substances diffuse into cells at any speed we 
please. 

Owing to a variety of means at our disposal cer- 
tain cells can be made to die in two minutes or in 
two hours, whichever one likes, merely by accelerating 
or delaying the diffusion of the agents into them; and 
it is clear that if a cell could control this diffusion it 
would at least make some effort to do so in order to 
save its own life. They cannot do so, however, and 
always die with clockwork-like regularity at the end of 
various given periods of time, which are determined by 
controlling the diffusion of certain chemical substances 
into the cell's cytoplasm. As will be shown later, the 
rate of diffusion of substances into living cells can be 
calculated by means of a simple equation; and since 
excitation, reproduction, and death can each in succes- 
sion be induced in vitro by causing the diffusion of 
substances into cells, it follows that excitation, repro- 
duction, and death may also be induced according to 
the rules which can be plotted as a simple equation. 



A PHYSICAL PHENOMENON 63 

Cells, as living entities, cannot refuse to absorb 
substances, and it is also a rule that they cannot "pick 
and choose" what they absorb. For instance, a cell 
cannot take from a solution which surrounds it a pro- 
tein and refuse an alkaloid. If it is surrounded by both 
these substances it has to take both. On theoretical 
grounds, I believe that a solution could be prepared 
(although we have not yet been able to assure our- 
selves that such is the case) from which a given cell 
would be able to absorb nothing; but such a contin- 
gency, as far as can be seen, would be impossible in the 
body. A cell does not appear to " feed " in the ordinary 
sense of the term — that is to say, it cannot seek after 
food. It has to take what is there according to certain 
laws, even if it dies in consequence; but it cannot 
"help itself" in any sense of the term. The life of a 
cell depends upon substances in its surroundings, even 
its reproduction depends upon them, and the associa- 
tion between them and its life depends on the diffusion 
of these substances into the cell itself, which diffusion 
is in its turn undoubtedly dependent on physical laws 
over which the cells themselves can individually exert 
no control. A cell cannot take a cake and leave a bun, 
so to speak: it has to take a bit of cake and a bit of 
bun whether it likes them both or not — a law which 
has been amply confirmed by work extending over a 
period of five years. 

One is open to criticism in this matter; for the 
objection may be raised that it is well known that 
some cells of the body are affected by some agents 
or drugs, while others apparently are not. This 



64 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

seems to be the case, judging from results; but it 
does not prove that all the other cells of the body 
have not also absorbed some of the drug in question 
as well, and that they may have been affected too, 
but have shown no signs of this effect. Strychnine, 
for example, stimulates certain cells of the nervous 
system, as is shown by the twitchings produced by 
it. Strychnine also causes amoeboid movements in 
leucocytes — a fact which is easily demonstrated micro- 
scopically; but this excitation of amoeboid movement 
within the body gives rise to no symptoms, and of 
course passes unnoticed. It is important to remember, 
therefore, that because a certain drug affects certain 
cells and gives rise to symptoms related to these cells, 
it does not follow that only those cells have absorbed 
the drug. All have absorbed a share, but not neces- 
sarily to the same extent, as will be shown directly. 

A cell, therefore, cannot control the diffusion of 
substances into itself; but after it has actually absorbed 
them the protoplasm of different classes of cells seems 
to treat a substance differently, and the cells may, by 
this peculiarity of their protoplasm, be able to make 
use of it, or, on the other hand, they may leave it 
unchanged, or thirdly, they may have to die from 
its effects. We shall presently describe how cells 
can be made to absorb aniline dye which contains 
two substances — one which causes the cell to re- 
produce itself, the other a poison which kills it. As 
both substances diffuse into the cell together, and 
as the cell cannot control this diffusion, it will respond 
to both. It will reproduce itself by cell-division in 



VERSUS "in vivo" 65 

response to one element, and it will die in the act 
of mitosis from the effects of the poisonous one. 
This experiment, which will be described at length 
later, proves these two points, about which I wish 
to be emphatic; viz. that a cell cannot control the 
diffusion of substances into itself, nor can it choose 
from its surroundings any one substance and leave 
another. Even at the expense of its life, a cell is 
bound to absorb from its surroundings any substance 
which may be present; and this absorption depends 
entirely upon certain chemical and physical factors. 

Before proceeding to describe these laws and 
factors, other points must be mentioned. We are 
dealing with in-vitro experimentation; and we have 
no proof that the diffusion of substances into cells 
in vitro is identical with this diffusion into cells 
in vivo. There is, however, strong presumptive evi- 
dence that similar conditions prevail. As a matter 
of fact, apart from the mere phenomenon of diffusion, 
this possible distinction between the facts learnt from 
in-vitro experimentation and what actually occurs 
in vivo must always be borne in mind in researches 
of this nature. The force of this point will become 
apparent later on when we come to deal with induced 
cell-division; for although one can induce the diffusion 
of substances into cells or cell-division at will on a 
microscope slide, it will be seen that these phenomena 
in the body occur under very different conditions, 
which must be taken into consideration in forming 
deductions from in-vitro experiments. In the final 
chapters, however, it will be shown that the results 

5 



66 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

obtained by in-vitro experimentation have been con- 
firmed in some instances by experimentation in the 
living body, and hence one may, I think, reasonably 
infer what goes on in vivo from what is observed 
in vitro, and that these experiments into individual 
cells may be undertaken with confidence. 

Before continuing this subject another matter con- 
nected with it must also be stated. In the previous 
chapter it was mentioned that cells observed in vitro 
must be resting in a solution or on a jelly which con- 
tains certain salts the presence of which are necessary 
for keeping them alive. In the body one of these salts, 
sodium chloride, is actually present; but there is no 
sodium citrate, a solution of which has proved to be 
the best one for leucocytes and other cells to live in. 
Obviously in the body there must be some salt or salts 
for which the sodium citrate is a substitute or equiva- 
lent. One of the roles played by sodium citrate in 
in-vitro experimentation is its property of preventing 
coagulation of the blood, which seems to be an im- 
portant one, for related to this is the curious fact that 
leucocytes will live longer in citrated plasma than in 
undiluted serum, a point which will be alluded to in 
the description of the method of measuring the lives of 
leucocytes. Sodium citrate, however, is detrimental to 
leucocytes, and there is no solution known which will 
keep leucocytes or other human cells alive for more 
than a few days. If there was we should now be in 
a position to cultivate families of human blood-cells in 
test-tubes. At present, by means of sodium citrate, 
one can only make leucocytes " exist" for some hours 



GAUGING THE DIFFUSION O/ 

while we experiment with them; and it must be borne 
in mind that since sodium citrate is detrimental, leuco- 
cytes or other cells placed in it gradually lose vitality 
all the time, and that they are under experimental 
conditions. 

The laws of diffusion — or rather what we know of 
them — are simple in their experimental application; 
but they are difficult to describe. 

There are two methods by which it may be known 
when substances have diffused into a cell. If the 
diffusing substance consists of a colouring matter which 
will combine with or otherwise colour the molecules of 
protoplasm within the cell, one can see the extent of the 
diffusion by watching the progress of the coloration. 
The other method consists in the use of a substance 
which has a specific action on the cell and causes it to 
give a definite response which will tell us when the 
substance has diffused in. Of the two methods, the 
former is obviously the better, for by seeing the gradual 
staining of the morphological elements of the cell one 
can more accurately gauge the extent of the diffusion 
than one can by measuring roughly the degree of a 
response such as excitation of amoeboid movements or 
even cell-division. It is, of course, possible to employ 
a combination of both methods, by which much can be 
learnt; in fact, in this book I shall describe what has 
been observed, in the first place, by using colouring 
substances only, afterwards a combination of stain and 
other substances, and lastly by experimenting with 
other substances by themselves. 

Suppose the jelly on which a given cell is resting 



68 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

contains a certain quantity of an aniline dye, such as 
Unna's polychrome methylene blue. This dye com- 
bines with the cell-granules and stains them red, and 
the rate of the diffusion of the dye can be estimated 
by observing the depth of coloration of the granules 
and the time occupied before the nucleus stains. The 
first granules to stain, of course, are those which are 
nearest to the jelly, for the cell is pressed against it by 
the cover-glass. With a given quantity of dye, the 
depth of coloration and the rapidity of the extent of 
staining will take a certain length of time. No matter 
how often this experiment is repeated, provided the 
arrangement of the jelly is always the same, with the 
same type of cell, the result is always the same; but 
if a fresh jelly is prepared, with double the quantity of 
stain, the depth of coloration will be double, and the 
same extent of staining will be reached doubly as 
quickly as with the first jelly. If the concentration 
of the dye is trebled or quadrupled, etc., the depth of 
coloration and the rapidity of the given extent of 
staining are also trebled, quadrupled, etc., as the case 
may be. 

Hence we arrive at the first law, which is, that the 
diffusion of a substance into a cell varies directly with 
the concentration of the substance in the solution in which 
the cell is resting. The more concentrated the sub- 
stance, the more it will diffuse into the cell, apparently 
in arithmetical proportion. In a given time, ceteris 
paribus, a 2-per-cent solution of a substance will have 
double the effect on a cell as compared with a 1 -per- 
cent solution. 



THE FACTORS COXCERNED 69 

Briefly, therefore, we may say that the diffusion is 
proportional to the amount of substance diffusing, or 
we may plot it thus: 

diff=S 

Obviously, the diffusion of a substance into a cell 
takes time. If there is only sufficient dye to combine 
with a certain amount of protoplasm, the combination 
will occur in a certain time, and then the diffusion will 
cease, for all the dye will be used up; but if there is 
a sufficiency of stain for it to go on diffusing indefi- 
nitely into the cell until it kills it by staining the 
nucleus, then the diffusion will go on for a longer 
time — in fact, it will go on diffusing minute after 
minute until death occurs. Hence we may say that 
the longer the time which we observe the diffusion, the 
greater will that diffusion be, unless the substance is 
all used up — a contingency which in reality cannot 
occur in practical experimentation, but it may occur 
in the body. It must be remembered that once the 
experimental jelly-film is made it cannot be altered, 
whereas in the body there can be no doubt that the 
solutions are being continually modified during meta- 
bolism. 

With a given concentration of dye or other sub- 
stances in the jelly, therefore, the greater the time 
during which the cell is resting on the jelly, the more 
of that substance will diffuse into the cell, also in 
direct arithmetical proportion. Each minute will see 
an equal amount of substance diffusing, provided the 
supply of that substance is constant, and that other 
conditions remain the same during the time. 



70 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

Conversely, in a given time, the greater the con- 
centration of the substance diffusing, the more of that 
substance will pass into the cell, as was shown in the 
first law. We have now considered two factors, there- 
fore, viz. that diffusion is equal to the concentration of 
the substance and the time, or thus: 

diff = S + T 

The next factor to be considered is heat. In vivo, 
of course, variations of temperature are not very great, 
but with in-vitro experimentation the temperature must 
be carefully considered, for we may keep the slide with 
its jelly-film with which we are working at a variety of 
temperatures, ranging from that of the room in which 
one works to that of the blood. Heat increases the 
diffusion of substances into cells in a marked degree, 
and this increase is also in arithmetical proportion. 
Each degree of temperature means a definite increase in 
the diffusion, and therefore the diffusion can be regu- 
lated to a nicety by keeping the slide on which the cells 
are resting at a definite temperature. Of course if 
extremes of heat are used death will occur; but within 
reasonable limits, which are compatible with life, one 
can employ heat to great advantage in these experi- 
ments. Heat therefore must be coupled with concentra- 
tion and time as a factor which increases diffusion ; and 
our equation now stands thus: 

diff=S+T+H 

There is one other factor which increases the diffusion 
of substances into cells more than any of the three other 



THE FACTORS CONCERNED 71 

factors already mentioned. Alkalies and alkaline salts 
greatly increase the diffusion of other substances into liv- 
ing cells. By means of a strong alkali one can cause a 
substance like stain to diffuse into a cell so rapidly as to 
induce death and staining of the nucleus almost instantly. 
And this marked increase of diffusion caused by alkalies 
also takes place in an arithmetical progression; that is 
to say, if the jelly or solution contains 2 per cent of an 
alkali, another substance present will diffuse into the 
cell twice as rapidly as it would if the jelly or solution 
only contained 1 per cent of the same alkali. Our 
equation must therefore contain a symbol for alkali 
also: 

diff = S+T + H + A 

All the above four factors — namely, the concentration 
of the substance diffusing, the time, the heat, and the 
alkalies — increase the diffusion. Neutral salts, however, 
decrease it. The more of a salt one adds to the jelly, 
the less of any other substance, ceteris paribus, will 
diffuse into the cell in a given time. And this retarding 
effect of a neutral salt also varies exactly with the 
amount of the salt present. We may therefore add 
salts to our equation with a minus sign before them, 
thus: 

diff = S+T + H+A-salts 

Acids, of course, delay the diffusion of other sub- 
stances, for they neutralise alkalies; and the amount of 
retarding effect due to an acid is in exact proportion to 
its neutralising effect on any alkali present. But apart 
from this neutralising action of acids, they also actually 



72 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

retard diffusion themselves according to their strength; 
that is to say, that if a certain amount of diffusion of a 
substance will occur from a neutral jelly, the addition of 
an acid will delay that diffusion in direct proportion to 
the amount of acid present. As a matter of fact, acids 
play only a very small part in these researches, for it 
has been our endeavor to copy the conditions found 
in the body as much as possible, and cells do not 
normally come into contact with acids to a great extent. 
For this reason, as will be shown later, we actually take 
steps to eliminate the consideration of acids from our 
experiments, in order to simplify matters. 

The foregoing, then, are the factors which increase 
or decrease the diffusion of substances into living cells. 
We have no right, of course, to assert that all alkalies 
increase and all salts retard the diffusion of substances 
into cells, for we have not tried them all; but as far as 
we have experimented they seem to obey a general 
rule. As has already been stated, one can only touch 
on the main principles of this subject of the passage of 
substances into individual cells, about which little was 
known before this jelly method of in-vitro staining 
was invented. 

Up to the present I have used the expression "cell" 
in its widest sense. Cells exist as individuals, and as 
individuals in classes. One may say that polynuclear 
neutrophile leucocytes are a class of cell, and that 
erythrocytes are another class of cell. 

The diffusion of substances into cells is generally 
the same in individuals of a class, but it presents great 
differences in the various classes. For instance, if a 



TAKES PLACE AT VARIOUS RATES 73 

jelly is suitably prepared to stain the nuclei of leuco- 
cytes in a given time, it will stain the nuclei of all 
the leucocytes in that time, and it will always do so. 
There will, of course, be a few exceptions among 
individual cells which have died or which have become 
achromatic, but generally speaking all the cells obey 
the rules of their class. In some classes of cells, how- 
ever, such as those of the epidermis, we have not yet 
succeeded in causing anything to diffuse into them at 
all; and in some of the larger cells, such as some 
epithelial cells, only a few types will absorb sub- 
stances in vitro; yet if some of the cells of a class in a 
specimen will absorb a substance at a certain rate, the 
others of the same class, which are not achromatic, 
will also absorb the substance at the same rate. It 
must therefore be grasped that the individual cells of 
a class will absorb substances in the same way as each 
other, and the diffusion into them will be influenced 
by the usual factors in the same way in each cell of the 
class; but substances diffuse into the cells of different 
classes at different rates. 

Now we come to an extremely important factor 
which has not been mentioned before, and which is the 
last one to be taken into consideration. It is the 
"coefficient of diffusion." 

We may prepare a jelly containing a certain con- 
centration of stain, alkali, and salts which will allow 
a certain amount of diffusion of the stain into a certain 
class of cells at a certain temperature in a certain 
number of minutes. Another class of cells may then 
be tried on a film made from the same jelly under 



74 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

the same conditions, when it may be found that now 
no staining, or less staining, may take place. If one 
adds more stain or more alkali, or more heat, or allows 
more time, this second class of cells may then stain. 
Hence we may say that the second class of cell has 
a higher "coefficient of diffusion" than the first, for 
it requires more of one or more factors which increase 
diffusion to cause a certain extent of diffusion into 
it than did the first class of cells. Different classes 
of cells may therefore each have different coefficients of 
diffusion, but in spite of this fact the diffusion of 
substances into all classes of them depends on the 
factors already expressed by the equation: 

diff = S + T ■+ H + A - salts. 

That is to say, that the factors given in the equation 
increase or decrease the diffusion of substances into 
all cells; bnt some classes of cells require more or 
less of them to cause the same amount of diffusion 
than do others. 

It is obvious, therefore, that we must always find 
the coefficient of diffusion of a class of cells before we 
can attempt to make substances diffuse into them; and 
we find the coefficient of diffusion by ascertaining the 
number of the factors expressed in the equation, and 
the amount of each of them required to cause a certain 
extent of staining of the cell. By means of the equa- 
tion we can set down algebraically the number of 
factors and the amount of each of them required to pro- 
duce this certain extent of staining; and then they are 



MODE OF DETERMINATION 75 

all added up to make a grand total figure — which repre- 
sents the "coefficient of diffusion," or, to express it 
briefly, the "c/" of the cell. 

The coefficient of diffusion of a cell is determined 
by adding up the total amounts of the factors required 
to cause a certain extent of staining of the cell. The 
extent of staining which we always use as a standard is 
the staining of the nucleus. Now, the "moment" of 
the staining of the nucleus of a cell can be recognized 
through the microscope, and it has an additional 
importance, insomuch as it is coincident with and 
signifies the death of the cell. In reality, therefore, the 
determination of the coefficient of diffusion of a cell, as 
well as supplying the rate of diffusion of substances into 
it, also tells us how much of the stain, together with 
the other associated factors, are required to make it (the 
stain) diffuse into the cell so as to cause the cell's death 
in vitro. In other words, it tells us the amount of a 
standard dye required to be in the immediate surround- 
ings of a cell, so that it may diffuse into it to such an 
extent as to cause its death by combining with the 
chromatin within the nucleus. 

In order to determine the coefficient of diffusion of 
a cell, however, it is necessary to count up, not only the 
number of the factors required to cause staining of the 
nucleus, but also the amount of each factor required. 
To do this it is necessary to measure each factor. One 
could, of course, measure the chemical factors, such as 
alkalies, salts, etc., in grammes, the heat in degrees of 
temperature, and the time in seconds; but this would 
necessitate a complicated total figure involving grammes, 



76 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

degrees, and seconds. It has been found preferable to 
measure these factors in special units which can, if 
necessary, be resolved into their -proper ones of 
grammes, degrees, and seconds. 

For instance, in order to remember the rate of stain- 
ing of a class of cells it would be most inconvenient 
to have to make a note of a statement such as this: 
To stain the nuclei in twenty minutes, it is necessary to 
keep the cells at 20° C. on a film made from a jelly con- 
taining . 5 cc. of Unna's stain, 0.16 gramme of sodium 
chloride, . 03 gramme of sodium citrate, and . 3 cc. of a 
5-per-cent solution of sodium bicarbonate. It is much 
simpler to say that the jelly contains so many "units" 
of stain, salts and alkali, heat and time. One may go 
farther and express these units as a simple equation, 
thus: 

Stain. Alkali. Heat. Time. Slats. 

Cy f = (5<?+ 3a + 3h+2t)-(c+2n). 

A letter by itself means one unit of the factor; a 
number before a letter means that there is that number 
of units of the factor: c means a unit of sodium citrate, 
3c would mean three units of it, and so on. 

It will be grasped that it is better to make "one 
unit" of any factor a standard quantity, and these 
quantities have been chosen with a special object. As 
has been previously explained, the coefficient of diffusion 
of a cell is the total number of units of the factors 
required to cause staining of the nucleus. Some of the 
factors increase the diffusion into the cell, and others 
decrease it. A unit of a factor which increases diffu- 
sion is so chosen that the increase it causes is equal to 



STANDARDISATION OF FACTORS 77 

that of one unit of any other factor which also increases 
diffusion. Likewise a unit of any factor which retards 
diffusion is also equal to a unit of any other factor 
which does the same thing. But further still, a unit 
which increases the diffusion of a substance into a cell 
is so chosen that the increase which it causes can be 
exactly neutralised by a unit of a factor which retards 
diffusion. The units are all equal in value, so to speak. 
Some increase diffusion, and some decrease it. Any 
number of units of factors which decrease diffusion 
retard exactly the increase of diffusion due to the 
same number of units of factors which cause increase of 
diffusion. 

By the first law, if we double the quantity of the 
dye in the jelly, we double the rapidity of its diffusion 
into the cells. A convenient quantity was chosen, 
namely, 0.1 cc, and this contained in 10 cc. of jelly 
constitutes one unit of polychrome dye. 1 Let us 
suppose that this quantity (one unit) causes staining 
of the nucleus of a given cell in a certain time. If 
now another unit is tried, the cell will stain in half the 
time it did before. 

The alkali, sodium bicarbonate, increases the dif- 
fusion of other substances into cells, and therefore it 
greatly increases the rapidity of the staining by poly- 
chrome methylene blue. Now, since all units must be 
equal in value, it was ascertained experimentally that 
0.1 cc. of a 5-per-cent solution of sodium bicarbonate 
exactly doubled the rapidity of diffusion of one unit of 

1 Unna's polychrome methylene blue (Griibler) is only supplied in 
solution, which is standardised. It cannot be made in a powder. 



78 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

polychrome dye. Hence the unit of alkali is . 1 cc. 
of a 5-per-cent solution of sodium bicarbonate. 

Time is a factor. One unit of time is ten minutes; 
and since time increases diffusion in arithmetical pro- 
portion, therefore in twenty minutes (two units) the 
diffusion of one or more units of the dye or other 
substance will be doubled. 

The unit of heat, is 5° C. ; unity is 10° C, because 
one cannot conveniently work at a temperature below 
this point; 20° C. is three units, etc. 

Salts delay diffusion. The two commonly employed 
are sodium citrate and sodium chloride. Their units 
respectively are . 03 gramme and . 08 gramme. One 
unit of sodium citrate or sodium chloride will prevent 
the increase of diffusion due to one unit of alkali, heat, 
or time; an extra unit of stain will neutralise a unit 
of salt. 

Hence the units of all the factors are so measured 
experimentally that they are as nearly as possible equal 
in value. The delay in diffusion caused by a unit of 
a substance which decreases diffusion is equal to the 
acceleration occasioned by one which increases diffusion. 
It will therefore be realised that the units can be 
substituted for each other. A unit of alkali will double 
the diffusion of the dye, but so will another unit of the 
dye itself. Again, the unit of time is ten minutes; 
since time increases the diffusion by arithmetical pro- 
gression, another ten minutes of time is equal to a unit 
of alkali or another unit of dye. Salts delay diffusion; 
we have found out how much of a salt, such as sodium 
citrate, is required to decrease this diffusion in equal 



EXPRESSED AS AN EQUATION 79 

proportion to the increase caused by one unit of alkali, 
dye, or time. All the units are equal to each other 
as regards the increase or decrease of diffusion, and 
therefore they are interchangeable. Hence we may 
simplify our equation by adding together all the units of 
all the factors and making a grand total of them ; thus : 

cf= (5s+3a + 3h + 2t) -(c + «») =13-3, 

or, simpler still: 

cf = 10. 

This method of determining the coeffieent of 
diffusion is intended principally to assist experimenta- 
tion with these in-vitro technics. The act of its 
determination gives up the comparative rate of the 
diffusion of other substances into the cells under 
observation, and tells us how to prepare jellies for 
further experimentation with these substances. For 
practical purposes, the equation and the measurements 
of the units of the several factors (which are used 
continually, not only in the initial determination of the 
coefficient of diffusion, but in all subsequent experi- 
mentation) have been devised with a view to the 
simplification of the practical methods to be described 
in the next chapter, where full details for the prepara- 
tion of the jellies, etc., will be stated. These laws of 
diffusion were ascertained in the first instance by me 
with the jelly method as described in the paper in the 
Journal of Physiology already referred to, and they 
soon led to the method of determining the coefficient 
of diffusion by the same method which was published 
in a paper in the Proceedings of the Royal Society 



80 DIFFUSION OF SUBSTANCES INTO LIVING CELLS 

(B. Vol. 81) ; and the description of the methods and 
laws given herein are in reality an elaboration of the 
original ones given in the papers mentioned. Much 
work has been done, however, since those papers were 
written, including induced cell-division by a variety 
of chemical substances, and all of it has been carried 
out with those specifications as bases. The point is 
mentioned in order to show that the method is fairly 
reliable. New technics of this nature, where one is 
dealing with a series of factors, all of which are 
variables, are prone to become involved in their 
application. The "moment" of the staining of the 
nucleus cannot be a very accurate one, and the more 
factors and units one deals with, the more do small 
errors assert themselves. 

It is a simple matter to note the effects on a cell 
of two or three units of a dye and a unit or two of 
alkali. But when one deals with complicated equa- 
tions involving twenty or thirty variable units, each 
of which modifies the action of its neighbour, it 
sometimes follows that complicated situations arise. 
For instance, the units of the two salts are satisfactory 
when small quantities of them are used; but with 
larger quantities it will be found that they are not 
quite so accurate. For practical purposes, however, 
the units given have been found to be sufficiently 
useful; but if this method was to be employed to 
determine the more scientific data of the action of 
the several physical factors in increasing and decreasing 
diffusion, I am prepared to admit that some units will 
require modification. 



CHAPTER VI 

THE PRACTICAL DETERMINATION OF THE ''COEFFICIENT 
OF DIFFUSION OF CELLS," AND ITS APPLICATION 
TO THIS IN- VITRO METHOD OF RESEARCH 

In the foregoing chapter I endeavoured to give an 
outline of the principles of diffusion of substance into 
individual cells, and the method of the determination 
of the coefficient of diffusion. In the present chapter 
I shall describe, in detail, how those principles are 
applied experimentally, and how one can find out 
the coefficient of diffusion of a given class of cells. 
The preparation of the jellies from which the films 
are made constitutes the most important part of the 
procedure. The chemical substances which are to be 
made to diffuse into the cells are contained in the 
jelly together with the other chemical factors, which 
increase or decrease diffusion. The factor heat is 
measured by keeping the slide on which the jelly-film 
is set at a certain temperature, and the length of time 
the slide is kept at this temperature determines the 
amount of the factor time. The coefficient of diffusion 
of a cell, as already pointed out, is arrived at by 

81 

6 



82 "coefficient of diffusion of cells" 

ascertaining experimentally the lowest sum of units 
of the factors — both chemical in the jelly and physical 
as applied to the slide — which will just cause the 
cell's nucleus to stain. In the original paper, already 
referred to, which specified this method and the co- 
efficients of diffusion, the following definitions were 
given : 

When a film of agar jelly contains stain and 
other substances, its Index of Diffusion (fx) may be 
defined as the sum of its constituents, which delay 
diffusion subtracted from the sum of its constituents 
which accelerate diffusion added to the quantity of 
stain contained in the jelly. 

The Coefficient of Diffusion (cf) of a cell is that 
Index of Diffusion plus the time and temperature 
required to cause staining of the nucleus, or staining 
of the cytoplasm in unnucleated cells (e.g. red cor- 
puscles), when the specimen is prepared by a standard 
method. 

It should be noted that the index of diffusion 
refers to the composition of the jelly, and that the 
coefficient of diffusion refers to the rate at which 
the cell absorbs substances from the jelly. 

The standard method of placing the cells on the 
jelly-film and the general principles of preparing the 
film have already been described. The cells are mixed 
with a little "citrate solution" (3-per-cent sodium 
citrate and 1-per-cent sodium chloride), which acts 
as a vehicle to keep them alive, and in which they 
are placed on the cover-glass. Since this citrate solu- 
tion spreads to the periphery of the cover-glass, it does 



"coefficient jelly" 83 

not materially influence the diffusion of the stain from 
the jelly into the cells. When experimenting with 
blood-cells the blood is mixed with an equal volume 
of the solution. In the case of other cells the mix- 
ture is made as may be convenient. In some instances, 
when the cells are naturally suspended in a fluid — 
such as pleuritic fluid — it is unnecessary to use any 
citrate solution at all, and the cells may be placed, 
suspended in their own fluid, straight on to the cover- 
glass. 

The general principle of preparing the jelly-film, 
as given in Chapter III., may be recalled. A 2-per- 
cent solution of agar in water forms a jelly basis for 
these experiments. This jelly is kept stored in large 
test-tubes, so that small quantities of it may be used 
without having to melt it in bulk everv time some 
is wanted; and it should be filtered when it is made 
in a manner similar to that employed for the prepara- 
tion of "nutrient agar," although, of course, it has 
no "nutrient" ingredients added to it. 

As already mentioned, the 2-per-cent solution of 
agar has such a consistency that it can, when melted, 
be diluted with an equal volume of a liquid and yet 
will set as a firm jelly on a slide when it cools. 

Experimentation with this method is essentially a 
process by which one contrasts the effects of one sub- 
stance on cells compared with those of others; hence it 
is important that all the conditions must be the same 
in each experiment, except the actual difference in the 
amount of the substance which has to pass into the 
cells. To this end the jelly-basis is always the same in 



84 "coefficient of diffusion of cells" 

every way, and each film is always made from a tube 
containing 10 cc. of this jelly. The substances which 
are to be tried on the cells are added to the jelly in the 
form of the Solution 2 (see Chapter III.)? which in its 
turn is added to the Solution 1. The combination 
(Solution 3) is always in the quantity of 10 cc, and the 
film is prepared from this. 

It has already been shown that the jelly-film must 
always contain certain quantities of the salts sodium 
citrate and sodium chloride, or the cells will not live on 
it. These salts are therefore added to the jelly-basis or 
Solution 1. They are added to it in bulk, so that any 
portion of it contains them, and, in consequence, it is in 
a condition to cause cells to live on it for as long as 
possible. 

The jelly is prepared as follows : 

In a beaker of water stand several of the large test- 
tubes which contain the stock 2-per-cent agar jelly. 
The amount required will be at least 50 cc. The 
water in the beaker should be heated until it boils, 
when the jelly in the test-tube will be melted. 

1 gramme of sodium citrate and then 0.8 gramme 
of sodium chloride should be weighed out accurately. 
The two salts are then placed in a flask, which should 
be of such a size that it also can be steeped in the 
beaker of boiling water; 49 cc. of the molten 2-per- 
cent agar solution from the test-tube are now measured 
out and poured into the flask. The salts slowly dis- 
solve in the molten agar, and, while this solution is 
taking place, the flask should be steeped in the boiling 
water in order to keep the jelly molten. 



"coefficient jelly" 85 

It is important that the sodium citrate should be 
neutral. Sodium citrate is inclined to become alkaline 
when exposed for long to the air, owing to the deposit 
of sodium carbonate. The jelly in the flask, therefore, 
must be tested and neutralised to litmus with citric 
acid. 

Previous to melting the jelly solution, a solution 
containing 8 . 3 per cent of citric acid should have been 
prepared; and now 1 cc. of that solution is added to 
the 49 cc. of the molten agar solution in the flask. 
This renders the whole of the jelly acid, the reason for 
which will be given directly. 

A series of ten clean test-tubes should be ready, and 
with a pipette 5 cc. of the acid jelly with its salts in 
solution is measured into each test-tube. Each of the 
ten test-tubes now contains 5 cc. of the jelly: total 
50 cc. in all. The 10 test-tubes are placed in a stand 
until the jelly is set, and a plug of wool is placed in 
each; otherwise moulds may grow on the jelly, as 
it contains salts. 

Every one of the 10 test-tubes contains 5 cc. of 
a 2-per-cent agar jelly, which is acid, because it contains 
in solution 0.0083 gramme of citric acid. It also 
contains . 1 gramme of sodium citrate and . 8 gramme 
of sodium chloride ; and these tubes of jelly are known 
for convenience as tubes of " coefficient jelly." 

To any one of these tubes we may add 5 more cc. 
of any solution or solutions; and if the whole is boiled 
and mixed by shaking, any portion of the 10 cc. of 
jelly mixture now contained in the test-tube will 
set on a slide as a firm jelly-film when it cools. 



86 COEFFICIENT OF DIFFUSION OF CELLS" 

Since it is essential that all jellies must be alike 
in all respects except in the actual quantities of the 
chemical substances which are to be tested on the cells, 
it follows that every jelly-film is always made from 
10 cc. of jelly. A film is never made direct from a tube 
of 5 cc. of "coefficient jelly" unless it previously has 
had added to it an equal quantity (5 cc.) of some 
solution. If this rule is followed, every jelly-film 
will be identical in that the strength of the agar will 
be the same, and the initial strength of the salts and 
acid will be the same; but since the second 5 cc. may 
be composed of any solution, each 10 cc. of jelly may 
also contain a variety of other substances. 

It is in the extra 5 cc. of solution or solutions that 
the chemical substances, with which one wishes to 
experiment on the cells, and any chemical factors 
additional to those already contained in the "coefficient 
jelly" which are required to increase or decrease dif- 
fusion, are added to that "coefficient jelly." 

The chemical factors, therefore, such as alkalies 
and salts, which increase or decrease diffusion assist 
to constitute the second 5 cc. of jelly which is always 
added to the 5 cc. of "coefficient jelly." Now, one 
could, of course, weigh out the right number of units 
of each factor for every experiment, but it is much 
simpler to add them from standard solutions. These 
standard solutions should be kept ready to hand in 
flasks, on the labels of which should appear the exact 
amount of each which constitutes one unit. 

The same may be said of the chemical substances 
the action of which one wishes to try on the cells. 



UNITS OF THE FACTORS 87 

For instance, in the determination of the coefficient 
of diffusion, the stain, as well as the alkali, is kept 
in standard solution, and is added to the 5 cc. of 
"coefficient jelly"; but it is most important to re- 
member that no matter how many units of each 
factor or substance may be contained in the 5 cc. of 
solution added to the 5 cc. of "coefficient jelly," the 
former solution must never be more nor less than 5 cc. 
Therefore, every jelly-film on the slide is always made 
from 10 cc. of jelly, which in its turn is composed of 5 cc. 
of " coefficient jelly" and 5 cc. of another solution bearing 
the units of the chemical factors. No matter how 
many units of no matter how many factors the second 
5 cc. of solution contains, it is always added in the 
quantity of 5 cc. — -no more and no less. Hence, the 
Solution 3, from which the film is prepared, will invari- 
ably consist of 10 cc. Solution 2 may contain one unit 
of one factor, or it may contain any number of units 
of any of the factors. 

If all the units of the contained factors exactly 
amount to 5 cc, all well and good; but if they do 
not do so, the balance must be made up to 5 cc. with 
water. By this means there will always be 10 cc. in 
the tube of jelly used for an experiment, but it may 
contain a great variety of units of the chemical factors 
which increase or decrease diffusion. 

The standard solutions of the several factors must 
be prepared with due regard to this rule. They must 
not be too dilute or their total may exceed 5 cc. The 
following list (abridged from the original paper on 
the "Coefficient of Diffusion") gives not only the actual 



88 "coefficient of diffusion of cells" 

units of the several factors, but also convenient standard 
solutions of them. It is useful to keep this list ready 
to hand in the laboratory. 

Alkali (sodium bicarbonate) increases diffusion. — 
Unit . 005 gramme. Standard solution 5 per cent, 
unity being 0.1 cc. It is convenient to remember 
that this solution is neutralised by a 4. 175-per-cent 
solution of citric acid, and that 1 unit of alkali is 
neutralised by . 1 cc. of such a solution. Since the 
agar at the outset is acid to the extent of . 083 gramme 
to 50 cc, a tube of 10 cc, made up as described, must 
contain . 0083 gramme of acid. This is exactly neutral- 
ised by . 2 cc. of the standard alkali solution ; that is, 
the agar at the outset, before any stain or other factor 
is added, delays diffusion to the extent of 2 units. 
Or, the addition of 2 units of sodium bicarbonate 
will render the agar neutral. 

Sodium Citrate delays diffusion. — Unit . 03 gramme. 
Standard solution 10 per cent, 0.3 cc. being unity. 
Since 50 cc. of agar contains 1 gramme at the outset, 
the 10 cc. of jelly may be said to contain about 3 units. 

Sodium Chloride delays diffusion. — Unit . 08 gramme. 
Standard solution 10 per cent, unity being 0.8 cc. The 
10 cc. of jelly contains this from the outset. 

Heat hastens diffusion. — Each unit 5° C. ; 10° C. is 
unity, 15° C. is 2 units, 20° C. 3 units, etc. For 
practical purposes I call 37° C. 7 units. 

Time increases diffusion. — Ten minutes is 1 unit, 
twenty minutes 2 units, and so on. 



REASOX FOR ACIDITY OF JELLY 89 

Stain, Unna's polychrome methylene blue (Grubler), 
behaves as if it increased diffusion. — Unit 0.1 cc. 

The reason why the "coefficient jelly" is made acid 
at the outset is this. Alkalies increase diffusion; 
acids delay it. Acids neutralise alkalies, and between 
the two there is a neutral point. If the jelly is 
neutral at the outset, we might have to add acid in 
the case of a cell having a very low coefficient of 
diffusion. Again, we may frequently have cases of 
cells which stain on a neutral jelly. Our equation, 
therefore, would have to include these three factors 
— alkalies, acids, and a neutral point — which would 
be very complicated, as the neutral point would 
introduce zero. The object throughout has been to 
make the determination of coefficient of diffusion of 
cells, and the estimation of diffusion of substances 
into them, as simple as possible in their practical 
application, and in order to do this the "coefficient 
jelly" is rendered acid at the outset and one deals 
only with the one factor — alkali. The original 50 cc. 
of jelly contains 0.083 gramme of citric acid; therefore 
each tube of 5 cc. of "coefficient jelly" contains 0.0083 
gramme of citric acid; and each tube will ultimately 
be made up to 10 cc, which, of course, will also 
contain this amount of citric acid, unless it is 
neutralised by alkali. This 0.083 gramme of citric 
acid represents 2 units of acid, and it is neutralised 
by 2 units of alkali. If we want to try a jelly which 
is acid to the extent of 2 units, we simply add no 
alkali. We are not likely to want a jelly which is 



90 COEFFICIENT OF DIFFUSION OF CELLS " 

more acid than this, for we have never yet seen 
any cell stain on a jelly which is acid beyond the 
extent of 1 unit. We must remember all along, 
however, that the jelly at the outset is acid to the 
extent of 2 units, and then go ahead with alkali. If 
we add to Solution No. 2 10 units of alkali, we say 
that the jelly contains 10 units; but in reality it is 
only alkaline to the extent of 8 units, for two of 
them have been utilised in neutralising the original 
2 units of acid. Of course, the neutralisation of 
the acid increases the content of sodium citrate to 
a slight extent, but it is so small that it can be 
neglected. As a matter of fact, by saying that the 
content of sodium citrate is usually 3 units, which is 
in excess of the reality, we compensate for the extra 
salt produced by neutralisation of the acid. The 
neutral point we ignore. If a jelly contains only 
2 units of alkali, it is in reality neutral; but we 
need not trouble about that. There is no neutral 
point in the equation, nor is there a symbol for acid; 
yet the neutral point and acid both exist in the 
equation, for the symbol "2a" means neutrality; 
and the symbol "a" means 1 unit of acid, whereas 
the absence of the symbol "a" means 2 units of acid. 
To recapitulate: Acids and the neutral point are 
omitted from the equation, but the jelly is acid at 
the outset, and we deal only with alkali. If a jelly 
contains only 2 units of alkali, that jelly is neutral. 
If a jelly contains 15 units of alkali, it really is only 
alkaline to the extent of 13 units. The jelly basis 
with which we work is known as "coefficient jelly." 



METHOD OF DETERMINATION 91 

It is kept in quantities of 5 cc. in test-tubes ready 
to hand. Each "coefficient jelly" contains sufficient 
salts for cells to live on it; it is acid to the extent 
of 2 units; and another 5 cc. of some solution must 
be added to it before it is poured on to a slide to make 
the "jelly-film." The film is always made from 10 cc. 
of jelly. 

In experimenting with a certain class of cells, 
one must in the first instance always estimate their 
coefficient of diffusion. The cells are mixed with 
"citrate solution" and kept ready at the room 
temperature, preferably in the revolving apparatus 
(see Chapter II.). 

In order to determine the coefficient of diffusion 
of these cells a tube of "coefficient jelly" is taken and 
a few units of stain are added to it, together with 
2 or 3 units of alkali solution. The content of the 
tube is then completed up to 10 cc. with water. The 
tube is steeped in the beaker of boiling water until the 
"coefficient jelly" all melts, when the stain and alkali 
become mixed with it, as will be presently described. 
A film is prepared from it on a slide, and a drop of the 
citrate solution, with the cells in suspension, placed on 
to it under a cover-glass. The specimen is kept at a 
certain temperature — representing a certain number of 
units of heat — until a certain number of minutes — repre- 
senting a certain number of units of time — have elapsed, 
and then the specimen is examined under the micro- 
scope. If the nuclei of the cells are not yet stained, a 
few more minutes (e. g. another unit of time — ten min- 
utes) are allowed. If, then, the nuclei are not stained, 



92 COEFFICIENT OF DIFFUSION OF CELLS " 

a fresh film is made from the same jelly, and it is kept 
at a higher temperature — or so many more units of 
heat — and again examined. If again it is found that the 
nuclei are not stained at the end of, say, half an hour, 
a fresh jelly is made, but with more units of alkali or 
stain, or both, added to a fresh Solution 2, which is 
added to a fresh tube of "coefficient jelly." If the 
nuclei still again remain unstained, one must try more 
units of time and more units of heat again. Thus we 
can go on trying fresh jellies, each of which contains 
more units of alkali, or of stain; and we try each jelly 
for a few minutes, first at a low temperature and then 
with a few more units of heat, until at last we find that 
the nuclei of the cells are just beginning to stain. The 
number of units of stain, alkali, heat, time, etc., of each 
film is noted on a piece of paper, and therefore there is 
no difficulty in knowing exactly how many units the 
jelly contained which was instrumental in staining the 
nuclei of the cells. The units of this jelly are then 
written out in the form of an equation, and those 
which retard diffusion — i.e. the units of the salts — 
are subtracted from those which increase diffusion, 
the difference being the number which is the co- 
efficient of diffusion of the class of cells experimented 
with. 

Examples. — -We wish to find the cf of the neutro- 
phil polynuclear leucocyte. A small quantity of 
citrate solution is drawn up into a capillary tube, as 
already described, and, the finger having been pricked 
and a small bead of blood squeezed out, an equal 
volume of blood is added to the citrate solution in 



EXAMPLES 93 

the capillary tube, which is placed in the "revolving 
apparatus." 

Take a test-tube of 5 cc. of "coefficient jelly," which 
of course, being cold, is set in the bottom of the tube. 
Add to it 0.4 cc. (4 units) of Unna's stain; 0.2 cc. 
(2 units) of alkali solution. Then the tube must have 
added to it 4.4 cc. of water, to make its total contents 
up to 10 cc. The colorless "coefficient jelly" will be 
set at the bottom of the tube, and above this will 
be 5 cc. of the mixture of stain, alkali, and water. 
The test-tube is then steeped in boiling water, when 
the jelly melts, and, as it does so, the stain, alkali, and 
water pervade the whole of its contents of 10 cc. In 
reality this 10 cc. of molten jelly is neutral, for the 
2 units of alkali have just neutralized the original 
acidity of the "coefficient jelly." When all is melted 
and mixed, the tube is taken out of the boiling water, 
and the contents are actually boiled, until they froth up 
in the tube, by holding the end of the tube in the flame 
for a few minutes. A drop of the boiling, stained 
mixture is then run on to the slide. Here it will set 
firmly in about three minutes, and if it is held up to 
the light the jelly-film will be found to be quite trans- 
lucent. A clean cover-glass is prepared, and a drop of 
the citrated blood is tapped out of the capillary tube on 
to it. The size of the drop is immaterial. The cover- 
glass is taken up between the finger and thumb, in- 
verted so that the drop of fluid is undermost, and it 
is allowed to fall flat on to the agar-film on the slide. 
The blood spreads over the film under the cover-glass, 
and the slide is then placed in the 37° C. incubator 



94 COEFFICIENT OF DIFFUSION OF CELLS 

(7 units of heat) for 10 minutes (1 unit of time). The 
index of diffusion of the jelly is this: 

where s = unit of stain, a = unit of alkali, c = unit of 
sodium citrate, and n = unit of sodium chloride. 

At the expiry of the ten minutes the specimen is 
examined, when it will be seen that the lymphocytes 
are quite unstained; but the granules of the polynu- 
clear leucocytes are just beginning to colour red. To 
find the cf of these cells, however, it is stipulated that 
their nuclei should just stain. The specimen is there- 
fore replaced into the incubator for a further ten 
minutes. Now it will be found that the nuclei of 
the eosinophile leucocytes are just staining. Hence, 
although this jelly has not yet given us the coefficient 
of diffusion of the neutrophile leucocyte, it has de- 
termined that of the eosinophile cell, which may be 
set down as follows : 

Eosinophile leucocytes 

cf = (4s + 2a + Ih + Zt) - (3c + n) = 1 1. 

where h = unit of heat and t = unit of time. 

In order to stain the nuclei of the neutrophile cells, 
we could place the same specimen for another ten 
minutes in the incubator; but it is not a very safe 
thing to do, for the cells by this time may be dead. 
It is better to make a fresh film from another jelly 
which contains more units of a factor which increases 
diffusion. We may add more stain or more alkali. 
Let us try another unit of each, thus: To a fresh tube 



EXAMPLES 95 

of 5 cc. of "coefficient jelly" add 5 units (0.5 cc.) of 
stain, 3 units of alkali (0 . 3 cc. of a 5-per-cent sodium 
bicarbonate solution), and 4.2 cc. of water to make 
it up to the 10 cc. of jelly. The film is prepared as 
before, and it is incubated at 37° C. for 10 minutes. 
On examination, it will be seen that the nuclei of the 
neutrophile cells are just turning scarlet. Hence this 
jelly at 37° C. in 10 minutes has the right Index of 
Diffusion for the coefficient of diffusion of neutrophile 
leucocytes. The equation may be thus set down: 

Neutrophile leucocytes 

C J = (5s + 3a + Ih +t) - (3c + n) = 12. 

The lymphocytes have a cf of 14 (2 units higher 
than that of the neutrophile cells). We may cause 
their nuclei 1 to stain in 10 minutes at 37° C. by using 
a jelly similar to the last one, but by either increasing 
the amount of alkali by 2 units, or by increasing the 
concentration of the stain by 2 units, or by increasing 
the alkali by 1 unit and the stain by 1 unit; or by 
increasing the time by 2 units. Let us try a jelly 
which contains 2 more units of stain, for now the 
chromatin of the cells will stain deeply and show up 
well. The jelly is made thus: To a tube of 5 cc. 
of "coefficient jelly" add 7 units (0.7 cc. of stain), 3 
units of alkali, and, since we now have more stain, 
only 4 cc. of water is needed to make the contents 
of the tube up to 10 cc. The whole mixture is boiled 
and a drop of it spread on a slide in the usual manner. 
After the blood has been mounted on it the slide is 

1 See Chapter XII. 



96 "coefficient of diffusion of cells" 

incubated at 37° C. for 10 minutes, when it will be 
seen that the nuclei of the lymphocytes have turned 
scarlet. 

Lymphocytes 

cf = (7s + 3a + 7h + t)-(3c + n)=U. 

In this specimen the neutrophile leukocytes will 
have burst, for the jelly has an index of diffusion too 
high for them by 2 units — their cf being 12. For 
the same reason the eosinophile cells will also be achro- 
matic, and the same may be said of the basophile cell, 
although it is very difficult to stain the nuclei of these 
cells. Their c/, however, is about the same as that of 
the neutrophile leucocyte. 

The simple equation has other advantages. It can 
be inverted, so to speak, and the units of the different 
factors can replace each other to some extent; for since 
the units of the several factors are equal to one another 
as regards their power of increasing or decreasing the 
diffusion, one can interchange them at will. We can 
make two jellies, for instance, one of which contains 
5 units of stain and 2 of alkali; and another which 
contains 2 units of stain and 5 of alkali; and provided 
the other factors are the same in the films made from 
each tube, the result obtained by each jelly will be 
identical. The equations will both give the same 
total : 

cf = (5s + 2a+4<h + 2t)-(3c + n)=g. 
cf = (2s + 5a + 4 i h + 2t)-(3c + n)=g. 

Any of the factors may thus be interchanged. 

We may delay this diffusion by adding more units 



REVERSING THE EQUATION 97 

of salts. The (Sc+n), however, is the usual content of 
salts which is always present in the "coefficient jelly, " 
but more salts may be added in the shape of solutions 
to the 5 cc. which also contains the stain and alkali. 
Whatever is added must be put down in the equation. 
The only substance not in the equation is agar, 
which, as already noted, does not affect the cells, and 
which is always present in the same strength in every 
experiment. 

Since the units of the factors are equal and inter- 
changeable, and since their sum is equivalent to the 
coefficient of diffusion, the numeral which constitutes 
the coefficient of diffusion in the equation can be inter- 
changed with an equivalent number of units of one 
or more of any of the factors. We may reverse the 
equation, therefore, and, provided we already know the 
coefficient of diffusion of the cell experimented with, 
we can, by this reversal, determine in a moment the exact 
quantity of any factor required to obtain staining of the 
nucleus. That is to say, that if the coefficient of diffusion 
is known, and if all the other factors, except one, are 
given quantities, then we can determine the required 
quantity of the one unknown factor simply by reversing 
the equation; always remembering the well-known 
algebraic law that in bringing one factor from one side 
of the equation to the other, we must change the signs: 
For instance, suppose a strain of Spirochceta refringens 
has a coefficient of diffusion of 20, and one wishes to 
stain a sample of them: Let us suppose there is a jelly 
to hand which contains 6 units of stain, 8 units of alkali, 
and the usual content of salts in the "coefficient jelly" 

7 



98 "coefficient of diffusion of cells 



>> 



from which it was prepared. The total contents of the 
tube has already been made up to 10 cc. as usual. The 
specimen is prepared and incubated at 37° C. Then the 
question must be asked, How long must the specimen 
remain in the incubator before the spirochetes will be 
stained ? We could, of course, keep taking the speci- 
men out and looking at it, but every time we did this 
we should lower the temperature and spoil the experi- 
ment. It is much simpler to plot the equation. The 
coefficient of diffusion of the spirochetes is a known 
quantity, i.e. 20; the time is now the unknown factor. 
We therefore exchange the places of the symbols cf 
and t , thus : 

t = (20cf + 3c+n)-(6s + $a + 7h)=3. 
t= 3, or 3 units of time, i.e. half an hour. 

Likewise, since the units of all the factors are equal, 
we may interchange any of them. Another example 
may be given. A certain strain of Amoeba coli from 
a "culture" has a cf of 13. We want to stain the 
nuclei of these parasites in 10 minutes with a jelly 
which contains 7 units of alkali. But we want to 
stain them at the room temperature of 20° C. The 
jelly contains its usual content of sodium citrate and 
sodium chloride — i.e. 3 units of the former and 1 of 
the latter. How much Unna's stain must we add to 
the tube of jelly to obtain the required result ? 

The number of units of s is the quantity required, 
hence : 

s = (13cf+3c + n)-(7a + 3h + t)=6, 



PRECAUTIONS 99 

six units of stain will be required, or 0.6 cc. of Unna's 
dye. 

The foregoing examples show how the coefficient of 
diffusion is determined, and how, after it has been 
ascertained, one can, by means of the equation, find 
out other factors, which may be unknown quantities. 
It follows that by this procedure other substances can 
be made to diffuse into the cells. This method of 
calculation has been used throughout these researches, 
and it will be seen that further examples will be given 
in the future chapters of this book. 

The factors most often employed are alkali and heat. 
Salts are not usually varied a greal deal, although their 
amounts can be altered if necessary by adding more of 
them to the second solution. 

The determination of the units of any other sub- 
stance is carried out on the principle that all units must 
be equal. Let us take a substance like urea, for in- 
stance. It delays the diffusion of other substances, 
such as Unna 's stain. All that has to be done is to 
find out how much urea must be contained in the 
10 cc. of jelly to neutralize the increasing action of a 
unit of alkali. Having found out the unit of the fresh 
substance, that unit is added to the equation in the 
usual way. If it increases diffusion it is placed in 
the bracket with the alkali and heat; if it delays diffu- 
sion it is bracketed with the salts. 

Lastly, having obtained the coefficient of diffusion 
of a class of cells by measuring the rate of diffusion 
of the stain, the stain may be omitted and any other 
substance substituted for it. If more than one sub- 



100 COEFFICIENT OF DIFFUSION OF CELLS 

stance is made to diffuse into the cells, they may each 
affect the diffusion of the other; for they may them- 
selves be alkalies, acids, or salts. In this case the unit 
of each must be found, and their action on the diffusion 
of other substances into the cell allowed for. 

It is necessary to point out that this method is 
reliable only within certain limits, and precautions must 
be taken to prevent errors due to employing excessive 
amounts of substances, heat, and time, and those due 
to possible contingencies arising when dealing with cells 
from tissues. 

The following list of precautions has been copied 
from the paper in the Proceedings of the Royal Society : 



Precautions. — As regards Life and Death: In a 
previous paper 1 it has been shown that the staining 
of the nuclei of leucocytes, when examined by this 
method, is a sign of death, and that the nuclei of dead 
cells will stain, ceteris paribus, before those of living 
cells. Consequently all the experiments given in the 
present paper have been made with fresh normal cells, 
and in the case of micro-organisms, with cultures not 
more than forty-eight hours old. It may also be men- 
tioned that the liquefaction of the cytoplasm which 
occurs after death materially alters the conditions of 
staining of leucocytes, and that the cf of living cells 
falls gradually after the blood has been shed. 

As regards Excess of Alkali, causing rapid death 
and liquefaction of the cytoplasm, with consequent 
prevention of staining (achromasia) : The addition of 

1 "On the Death of Leucocytes" (H. C. Ross, Journal of Physiology ,. 
vol. xxxvii., p. 327,1908). 



PRECAUTIONS 101 

excess of alkali may cause death, staining of the nuclei, 
liquefaction, and the loss of stain on the part of the 
cells. 1 This may occur before a preparation can be 
focused, in which case the cells appear unstained 
and will refuse to stain, no matter how much stain or 
alkali are tried. Therefore it is better to begin with a 
low index of diffusion and to try tube after tube, each 
containing a little more alkali, until staining is ob- 
tained. Further, the amount of sodium bicarbonate 
should not exceed twenty units, because, as has already 
been pointed out in a former paper, 1 if added to excess 
it may act as a neutral salt and delay diffusion. 

As regards Excess of Deficiency or Heat: A tem- 
perature above 40° C. may allow the cells to diffuse 
through the agar. 2 A temperature below 15 degrees 
has not been experimented with, because, even at a 
temperature of 20° C. it requires a minimum of 3 units 
of stain to cause staining of the nuclei of leucocytes 
in spite of the addition of a large amount of alkali, 
for the alkali is not sufficient, per se, to cause the 
cells to absorb sufficient stain to colour the nuclei 
unless the stain is concentrated. 

As regards Excess of Time: A period of more 
than half an hour has not been employed for fear 
of death and liquefaction of the cytoplasm, for the 
cells may die and become achromatic before there 
has been time for sufficient stain to diffuse into them 
to cause staining of the nuclei, in which case, of course, 
the cells will never stain. 

As regards Excess of Stain: More than 10 units 
of stain may cause precipitation of the agar as the 
film cools on the slide, and the precipitate carries 

1 "On the Death of Leucocytes" (H. C. Ross, Journal of Physiology, 
vol. xxxvii., p. 327, 1908). 

"The diffusions of Red Blood Corpuscles through Solid Nutrient Agar" 
(H. C. Ross, British MedicalJournal, May 5, 1906). 



102 COEFFICIENT OF DIFFUSION OF CELLS 

some of the stain down with it, vitiating the results, 
for it has been shown that agar is not very soluble 
in cold stain. 1 

As regards Examination: The observation of cells 
floating near a bubble under the cover-glass should 
be avoided. The fact that blood-cells in such a 
situation will stain before others has already been 
noted. 1 I consider this to be due to these cells floating 
in a small quantity of alkaline citrated plasma collected 
round the bubble. 

Consequently the experiments have all been made 
within the compass of the above restrictions. It may 
also be advised that when unnucleated cells contain 
granules in their cytoplasm the staining of the gran- 
ules gives a more constant rate than the staining of the 
cytoplasm. By this means it is seen that the cf of the 
blood-platelet is identical with that of the polymorpho- 
nuclear cells. 

1 "On the Death of Leucocytes" (H. C. Ross, Journal of Physiology, 
vol. xxxvii., p. 327, 1908). 



CHAPTER VII 

DIFFUSION OF SUBSTANCE INTO CELLS TO EXCESS 

DIFFUSION- VACUOLES OR " RED SPOTS " THE PROOF 

THAT THE BLOOD-PLATELET IS A LIVING CELL 

In this chapter I shall discuss the effects of the diffusion 
of substances into a cell, when that diffusion occurs to 
excess. A cell's protoplasm can utilize only a certain 
amount of the dissolved substance or substances which 
diffuse into it from the immediate neighbourhood of 
the cell. One can, however, push this diffusion by the 
agency of one or both of the factors — heat and alkali 
— which increase diffusion, and if we do this some of 
the neighbouring liquid itself passes into the cell and 
remains suspended as minute droplets in the cytoplasm. 
These droplets have been called "diffusion- vacuoles.' ' 
When they were first seen, five years ago, the cells were 
resting on stained jelly,'and since the "diffusion- vacuoles" 
were stained they were therefore called "red spots." 

Diffusion-vacuoles must not be confounded with 
the ordinary vacuoles (fig. 16) which appear as colour- 
less, non-granular patches in leucocytes. Many theories 
have been advanced regarding these latter vacuoles, 
but although we have so often seen them, we have no 
explanation to offer as to their nature. They are 

103 



104 DIFFUSION- VACUOLES 

certainly not composed of liquid; they are not cavities; 
and, so far as we have observed, they play no part in 
cell-division. When the cytoplasm liquefies at death 
they disappear, and when a cell divides they seem to 
migrate into the cytoplasm, remaining outside the 
chromosomes and centrosomes. 

The diffusion-vacuole is quite another kind of body 
(fig. 17). It is never seen in a normal cell which has 
been freshly removed from the tissues. "Red spots" 
always appear gradually (fig. 18), beginning as minute 
coloured points in the cytoplasm, which gradually 
become larger until — in the case of leucocytes — they 
may become as large as a lobe of the nucleus. Two 
or more may coalesce to form one large diffusion- 
vacuole; and their appearance depends entirely on the 
laws of diffusion; in fact, they may be produced in 
leucocytes at will by arranging the plus factors, heat 
and alkali, in the equation in such a way that they 
promote the diffusion of a substance to excess. 

Diffusion-vacuoles appear only in living protoplasm. 
After death the cytoplasm liquefies and the cell 
becomes disorganized, when diffusion-vacuoles cannot 
appear in it. The actual passage of a substance, say, 
stain, through a living cell's cytoplasm occupies a 
certain amount of time, which can be shortened by 
heat or alkalies and lengthened by salts. If heat and 
alkali are present, but the salts are absent, the stain 
diffuses into the cell so quickly that death may ensue 
in a few moments, because the nucleus becomes stained. 
Indeed, one may thus cause death in a few seconds; 
and death is accompanied by liquefaction of the cyto- 



THEIR NATURE 



105 




Fig. 16. — A stained leucocyte. The ordinary vacuoles (colourless patches 
amongst the cell-granules) are well shown. The cell has just died. 




Fig. 17. — Diffusion-vacuoles in a leucocyte. 



THEIR NATURE 107 

plasm, which, when it is alive, appears to be in the form 
of a jelly. Now, it is obvious that if the cytoplasm 
liquefies in a few seconds, diffusion-vacuoles cannot ap- 
pear, for it is unlikely that a liquid like a solution of stain 
cannot remain suspended in droplets in another liquid 
like liquefied cytoplasm. On the other hand, if the 
cytoplasm is alive and jelly-like, any excess of stain which 
diffuses into it will become suspended in it as a "red 
spot." Hence, if death is caused extremely rapidly, no 
matter to what excess the diffusion is increased, diffusion- 
vacuoles will not appear, and, owing to the excess, 
liquid passes into the cell. If this excess is great, the 
dead cell will be seen to burst (it appears even to 
explode sometimes, especially if there are no salts to 
delay the diffusion), and the cell-granules are scattered 
about the field of the microscope. It is a well-known 
fact that if water is mixed with blood, the leucocytes 
will burst, the reason being the same, for the water 
passes into the killed and liquefying cytoplasm, and the 
intracellular tension is so great that rupture of the cell- 
wall takes place. There are no salts to delay the 
diffusion of the water, which now occurs to such excess 
that it causes the cell to rupture. 

In order to demonstrate the diffusion-vacuoles, 
therefore, it is necessary to delay death, which can 
be done by placing salts in the jelly-film such as are 
present in the "coefficient jelly." Diffusion is then 
increased by alkali or heat until maximum diffusion, 
short of causing death, is obtained; for it must be 
remembered that all artificial substances will kill 
human cells, and the more they diffuse into them the 



108 DIFFUSION- VACUOLES 

more rapidly will the cells die. If now half a unit 
more of alkali is added to the jelly, or two or three 
more degrees of temperature are tried, diffusion- 
vacuoles will gradually make their appearance in all 
the cells. 

For instance, "red spots" are readily produced 
in leucocytes. Any jelly which has the correct index 
of diffusion for a coefficient of diffusion of 12 will 
cause them to appear if another drop of alkali is 
added to the jelly. The diffusion of the stain must 
be excessive; but not so excessive as to cause death 
in a few seconds. It is necessary to hit off those 
amounts of alkali and heat which will cause liquid 
to pass into the cells, but which will not unduly 
hasten death by staining the nucleus too rapidly. 
If this is done accurately, these remarkable diffusion 
vacuoles suddenly begin to appear. If the diffusion 
is still further increased, the cells will burst and 
become achromatic instantly. The appearance of 
the diffusion-vacuole may be regarded as the safety- 
point of diffusion; and it is a signal that no more 
alkali or heat may be tried, or the cells may burst. 

It is interesting to watch the fate of these vacuoles. 
Since the substance is diffusing into the cells to excess, 
this diffusion must cause death in a short time, 
even though the cells do not burst. Before this 
occurs, however, the diffusion steadily increases, and 
the "red spots" get larger. When death takes place 
the cytoplasm liquefies slowly, beginning at the 
periphery and progressing more and more towards 
the nucleus. As the cytoplasm liquefies, more and 



DISPERSAL OF THE VACUOLES 



109 




Fig. 18. — A dead leucocyte in which diffusion-vacuoles are beginning to 

appear. 




Fig. 19. — A ditiusion-vacuole in a lymphocyte. Low power. 



DISPERSAL OF THE VACUOLES 111 

more of the cell-granules show the remarkable danc- 
ing Brownian movements, and the liquefying cyto- 
plasm gradually involves the diffusion-vacuoles, one 
by one. When the liquefying cytoplasm, which 
immediately surrounds a vacuole, becomes of the 
same consistency as the liquid within the vacuole, 
the latter, which in reality is like a bubble of liquid 
suspended in a liquefying jelly, suddenly bursts and 
disperses, leaving a cup-shaped cavity in that portion 
of the more central cytoplasm which has not yet 
become liquefied. One by one all the vacuoles 
disperse, and either immediately before or after 
their dispersal general achromasia of the cell ensues, 
for achromasia also depends on the liquefaction of 
the cytoplasm. Vacuoles have never been seen to 
"disperse" in a living cell; it is necessary for the 
cytoplasm to liquefy for this to happen, and lique- 
faction occurs only at death. Diffusion- vacuoles will 
frequently be seen when experimenting with this 
in-vitro method, large numbers of them sometimes 
making their appearance in a single cell; but they 
will all disappear after a short time. I have seen 
them in all varieties of blood-cells (figs. 19, 20). 

The colour of the diffusion-vacuole depends on the 
colour of the solution or jelly in which the cell is 
resting. If the jelly contains a red dye, such as 
polychrome blue, the vacuoles will be red; ordinary 
methylene blue causes them to appear blue. If no 
stain is present the vacuoles will be colorless; if 
stain is present the coloration of a vacuole is always 
deeper than the colour of the surrounding jelly. We 



112 DIFFUSION- VACUOLES 

believe the reason for this is, that when the droplet of 
liquid becomes suspended in the jelly-like cytoplasm 
it forms a cavity in it, and the walls of the cavity 
actually become stained. This is readily seen when 
the vacuoles disperse, for portions of the stained wall 
of the cavity can be demonstrated. When cytoplasm 
is wounded (the formation of a vacuole in it really con- 
stitutes a wound of it) the cytoplasm stains deeply with 
an anilin dye, and this appears to be the reason why the 
"red spots" seem to be so deeply coloured. Moreover, 
being spherical droplets, they are highly refractile. 

We have never seen diffusion-vacuoles in normal 
cells immediately after they have been removed from 
the body; it is always necessary to induce them. 
There is an exception to this rule, however, in the 
cells of some malignant growths, especially cancer of 
the breast, in which we have frequently seen large 
"red spots." We think that it is possible that the 
injured cytoplasm associated with these spots may 
produce the deeply staining patches which have been 
described as "archoplasm" in these cells when they 
are fixed and stained by the older methods. In a 
former paper we also suggested that archoplasm might 
be derived from chromatin which has diffused through 
the cytoplasm to some extent, and we still think that 
this may be possible, but it is also probable that archo- 
plasm is derived from the fixation of injured cytoplasm 
connected with a diffusion-vacuole. We have never 
seen anything like the commonly described archoplasm 
in a normal living leucocyte, and it certainty does not 
play any role in their cell-division. 



CAUSED BY A LOWERED COEFFICIENT 113 

There appears to be little doubt that archoplasm 
does not exist normally in a living cell; it can be 
produced in them, however, by lowering their co- 
efficient of diffusion by keeping them for some hours 
in extracts of dead tissues — and this is, we believe, the 
reason why it appears so frequently in living cancer- 
cells. 

It is obvious that substances will, ceteris paribus, 
diffuse more readily into a cell if it has a coefficient 
of diffusion lower than its normal one, and, for the 
same reason, vacuoles can more easily be induced in 
it. For instance, no diffusion-vacuoles will appear in 
fresh leucocytes when they are resting on a jelly which 
will not cause the maximum diffusion of stain into 
them; but if we lower their coefficient of diffusion, 
and again place them on the same jelly, not only may 
the maximum amount of stain now pass in, but it may 
pass in to excess, and diffusion-vacuoles will appear. 
This fact has led to the determination of a point of 
scientific interest which has been controversial for more 
than half a century. It has proved that the blood- 
platelet is a living cell 1 ; for diffusion-vacuoles will 
not appear in the normal blood-platelets, but if their 
coefficient of diffusion is lowered by causing gradual 
death, the lowering of the coefficient of diffusion so 
occasioned will now permit "red spots" to be induced 
in them. 

Our experiments up to the present have revealed 
the fact that the coefficients of diffusion of all cells 
fall gradually as their vitality fails, provided this loss 
of vitality is gradual. The coefficient of diffusion of 

l A paper by myself on "V. Ph. Vacuolation of Blood-platelets" was 
published in The Proceedings of the Royal Society, B., vol. lxxxi, 1909. 



114 DIFFUSION- VACUOLES 

leucocytes may fall by as much as one unit if the cells 
have been shed for more than twenty-four hours and 
kept in citrate solution at the room-temperature. 
There are certain substances also which expedite this 
loss of vitality and its accompanying lowering of the 
coefficient of diffusion, especially certain alkaloids and 
extracts of dead tissues; and it was in the course of 
experimentation with the alkaloid morphine hydro- 
chloride that diffusion-vacuoles were seen in the 
blood-platelets for the first time. 

The events which led to the discovery of diffusion- 
vacuoles in blood-platelets are worthy of mention. 

Soon after this method of in-vitro staining was 
suggested by Professor Ronald Ross about five years 
ago, either he or I saw the "red spots" for the first 
time in leucocytes. The laws of diffusion which I 
have described were not then known, and only minute 
vacuoles had been seen in the cells, for alkalies had 
not been employed. These spots only appeared as 
minute red points in the cytoplasm, and in appear- 
ance they certainly resembled the centrosomes of 
plants and other cells; for it must be remembered 
that hitherto leucocytes have never been seen to divide, 
and no one knew what their centrosomes were like. 
In the preliminary note in The Lancet 1 by Professor 
Ross and Messrs. Moore and Walker, in which this 
in-vitro method was first described, these "red spots" 
were mentioned, and it was suggested that, from their 
appearance, they might possibly be centrosomes. Now, 
it is well known that the nature of the blood-platelet 

l The Lancet, July 27, 1907. 



NATURE OF BLOOD-PLATELETS 115 






Fig. 20. — A diffusion-vacuole in a granular red cell. 



A 



Fig. 21. — A clump of normal blood-platelets. They are resting on a jelly 
which will just stain their granules. 



NATURE OF BLOOD-PLATELETS 117 

(fig. 21) has been a matter of great controversy for many 
years ; some say that they are normal constituents of 
the blood, but are precipitates of the plasma. Others 
say that they are extruded nuclei of red cells, and 
again it has been suggested that they are derived from 
leucocytes. Lastly, some people say, even to this day, 
that they arise from all three sources. In view of this 
controversy, Professor Ross and his collaborators, con- 
sidering the "red spots" in leucocytes to be centro- 
somes, suggested that if anybody could find them in the 
blood-platelets it would, of course, settle once and for 
all the real cellular nature of these bodies. 

A short time after this, while I was working to 
determine the laws of diffusion by this method, I 
appreciated that "red spots" were not centrosomes at 
all, but were diffusion-vacuoles — a fact which I pub- 
lished in The Journal of Physiology, 1 and a fact which 
was afterwards confirmed when divisions were induced 
in leucocytes and the real centrosomes demonstrated. 

This knowledge rendered Professor Ross's sugges- 
tion of less importance, for since the spots are not 
centrosomes, the discovery of them in the platelets 
would not prove that these bodies found in the blood 
were cells capable of reproduction. But when I was 
experimenting with morphia on blood-cells I acci- 
dentally discovered the "red spots" in all the blood- 
platelets (figs. 22, 23). 

Now, in spite of the fact that these spots are 
not centrosomes, their appearance in the blood-plate- 
lets still proves that these minute bodies are living 

1 Journal of Physiology , vol. xxxvii, No. 4. 



118 DIFFUSION- VACUOLES 

cells; because these diffusion- vacuoles will appear only 
in living cytoplasm. 

Moreover, vacuoles will never appear in normal 
blood-platelets — they are never seen in fresh blood- 
films. It is necessary to keep the blood in an equal 
volume of citrated solution of morphia (a 1-per-cent 
solution of morphine hydrochloride in citrate solution) 
for four hours at 37° C. A drop of the mixture is then 
examined on a film of jelly in the usual way, and the 
film of jelly should have the correct index of diffusion, 
and be kept at the right temperature and time for 
the coefficient of diffusion of leucocytes, i.e. 12. The 
diffusion-vacuoles will then appear in all the blood- 
platelets. This experiment is a very easy one, and 
certain in its results. 

The action of the morphia is the same on the 
blood-platelets as it is on leucocytes and lympho- 
cytes. It lowers the coefficient of diffusion to a 
marked degree, and it appears to do this by causing 
gradual death. Morphia, in the 1-per-cent solution, 
is a slow poison for leucocytes, for it will kill most 
of them at 37° C. in about six hours. After incuba- 
tion for four hours, however, when the cells are placed 
on the jelly, the cells are still alive, but their coeffi- 
cient of diffusion is so lowered by the poison that 
the jelly, instead of merely causing maximum diffusion, 
now causes diffusion to excess, and the leucocytes 
and lymphocytes become intensely vacuolated (fig. 
24). Further, the blood-platelets will now exhibit 
"red spots." 

In addition to lowering the coefficient of diffusion 



ARCHOPLASM 



119 





Fig. 22. — Diffusion-vacuoles in blood-platelets. The cells are resting 
on the same jelly- film as those in 21, but they had been subjected to the 
action of morphine hydrochloride. 




' W 




Fig. 23. — Diffusion-vacuoles in blood-platelets. The jelly-film had the 
same index of diffusion as that employed in 21. 



ARCHOPLASM 



121 




Fig. 24. — A specimen of blood which had been mixed with morphia 
solution. Note the extreme vacuolation of the leucocyte. A blood-platelet 
is also vacuolated. The same jelly as in 21. 




k 1 

Fig. 25. — Patches resembling archoplasm induced in a leucocyte by 
subjecting the blood to an extract of a dead tissue. The jelly-film on which 
the cells are resting is similar to that employed in 21. 



ARCHOPLASM 123 

by causing gradual death, morphia undoubtedly has 
a profound effect on the cellular cytoplasm, for other 
alkaloids, so far as they have been tried, do not cause 
vacuolation of the platelets. On the other hand, 
we have occasionally seen a vacuolated blood-platelet 
from a specimen of blood which has been mixed for 
about twelve hours with a citrated solution (100 per 
cent of suprarenal extract). It has already been 
mentioned that extracts of dead tissues lower the 
coefficient of diffusion, and in producing vacuolation 
they also produce archoplasm in leucocytes (fig. 24). 
Possibly, as mentioned before, the archoplasm which 
is so frequently seen in cancer cells is derived from 
the vacuolation caused by the action of the remains 
of dead tissues on the cells. If leucocytes which have 
been subjected to morphia and have been placed on 
jelly as above described are watched for some time, 
patches which might be described as archoplasm may 
often be seen in them as a result of the dispersal of 
many of the diffusion- vacuoles induced by the alkaloid. 
We cannot, of course, state definitely that these patches 
are identical with what is known as archoplasm, and 
we have never seen anything which could be described 
as it in normal leucocytes examined by this method; 
but that induced in them by extracts and morphia is 
nearer the usual interpretation of archoplasm as seen 
in fixed specimens than anything we have seen. 

Since the blood-platelets can be made to become 
vacuolated by lowering their coefficient of diffusion 
by the action of the poison morphia; and since all the 
blood-platelets in a specimen thus respond to it, it 



124 DIFFUSION- VACUOLES 

is clear that the blood-platelet is a living cell and 
not a precipitate. As far as we know, no precipitate 
has a coefficient of diffusion, and even if such a 
thing were possible, one certainly could not lower it 
by causing approaching death with morphia. 

Blood-platelets unquestionably are living cells; and 
they can actually be seen to be produced by leuco- 
cytes when they are examined on a jelly-film by 
this method. They are all the same class of cell, ap- 
parently produced in the same manner. If a speci- 
men of fresh blood is spread on a jelly which contains 
stain and an alkaloid such as atropine sulphate, as 
will be described in the next chapter, the leucocytes 
and the lymphocytes become excited and extrude long 
pseudopodia. Sometimes these pseudopodia become 
detached from the cells (fig. 26), when the fragment 
appears to be identical with a blood-platelet. They 
may contain a few granules derived from the leucocytes. 
Moreover, the blood-platelet is also highly amoeboid 
under this excitation; and their amoeboid movements 
can easily be seen by this method. Deetjen, several 
years ago, asserted that blood-platelets showed amoeboid 
movements, although, of course, he did not employ 
alkaloids to excite them. By this method, however, 
not only can they be readily seen to show movements, 
but they have also actually been photographed in the 
act (fig. 27). We have also succeeded in obtaining 
a negative of a blood-platelet apparently being produced 
by a leucocyte (fig. 26) . As a matter of fact, the plate- 
lets stained by this method have such a remarkable 
resemblance to leucocytes that in the very earliest 



NUCLEATED RED CELLS 



125 





Fig. 26 — An extruded pseudopodium becoming detached from a leucocyte 
which is excited by atropine. No stain. 




Fig. 27. — Amoeboid movements excited in a blood-platelet by the action 

of atropine. 



NUCLEATED RED CELLS 127 

experiments it became apparent that these bodies were 
associated with those cells. We have never succeeded, 
however, in making a platelet reproduce itself, even with 
the most powerful exciter of cell-division. Blood-plate- 
lets frequently become clumped into masses, especially 
if the jelly contains an extract of a tissue; we therefore 
think that this clumping may have some function in the 
phenomenon of healing. 

At this juncture it may be useful to dispose of an 
old theory, that the blood-platelet is the "extruded 
nucleus" of a red cell. In the first place, no diffusion- 
vacuoles have ever been seen within the nucleus of any 
cell, and the platelets, therefore, can hardly be nuclei. 
This suggestion of the nuclear origin of the platelet 
would never have occurred, I think, if the originators 
of it had used in-vitro staining. Red cells are never 
seen to extrude their nuclei by this method as they 
sometimes seem to be in the act of doing when they 
are spread out and fixed on a slide by the old methods- 
It is difficult to imagine that any cell could extrude its 
nucleus bodily, and a glance at the stained nucleus of 
an unfixed nucleated red cell will dismiss such a fallacy 
in very short time. The nucleus of a living red cell 
seems to consist merely of a mass of chromatin granules, 
which appear to be identical with those of the "red 
cell with basic granules." The granules ultimately 
seem to disappear altogether, for in normal blood one 
sees about 1 per cent, of these granular cells, which 
sometimes have only one or two granules, whereas in 
anaemia the number of granules is much greater in 
most of the cells. Presumably, when the granules 



128 DIFFUSION- VACUOLES 

have disappeared altogether the cell resembles the 
ordinary erythrocyte. The nucleated red cell has a 
coefficient of diffusion of about 11, and so has the 
granular cell. An ordinary erythrocyte's coefficient of 
diffusion seems to be much higher, however; but since 
it has no nucleus or granules to stain, it is difficult to 
determine it. 

To stain the stroma of an ordinary red cell it re- 
quires a jelly with an index of diffusion of nearly 20. 
Like other blood-cells, the coefficient of diffusion of 
red cells falls the longer the blood has been shed, 
until, with a jelly suitable for staining the nuclei of 
leucocytes, the stroma (or perhaps it is the haemoglobin 
itself) of red cells will stain in many instances. It 
is presumed that this more rapid staining of the stroma 
or haemoglobin of red cells which have been shed some 
time is due, as in other cells, to the lowering of the 
coefficient of diffusion, for extracts of dead tissues and 
morphia also have this effect on them. 

The stroma or haemoglobin — whichever it may be — 
stains more readily in nucleated and granular red cells 
than in the others. "Red spots" will fairly often be 
seen in nucleated red cells and in granular ones; but 
they have only been seen three times in ordinary 
erythrocytes. 

Apart from their scientific interest, however, diffu- 
sion-vacuoles are not of great importance, we think, 
except that their appearance, as noted above, is a 
signal that maximum diffusion is being occasioned. 

I have now described what we know concerning the 
diffusion of substance into living cells. It is a complex 



FACTORS ACT ON THE CELLS 129 

subject, which will require careful elucidation if the 
actual physical laws on which it is based are to be 
found out, and I venture to think that this method will 
supply a means by which these laws can be determined ; 
a large amount of careful experimentation will be 
necessary, however, with a large variety of substances. 
The chemical factors, such as alkalies and salts, will 
have to be tried in greater variety; after which it 
seems to me probable that one will be able to settle 
whether the increase and decrease in diffusion which 
they cause is due to their atomic weight or the osmotic 
pressure, or what. One point, however, should be 
clearly appreciated, which is this, that these chemical 
factors which increase or retard the diffusion of other 
substances, act not on the substance diffusing into the 
cell, but on the cell itself. For instance, as will be 
shown later on, alkalies, by increasing the diffusion 
of kreatin or xanthin, increase the rapidity of cell- 
division induced by these extractives. But the alkalies 
have no effect on either kreatin or xanthin. The way 
they increase diffusion into the cell is by causing the 
cell to absorb substances more readily. And so with 
acids, salts, and other chemical factors. 

Lastly, these simple laws of diffusion must be taken 
into consideration throughout researches with this 
method, for no results will be obtained if they are 
forgotten. The equation has been found to be of 
more use when stain is employed. Later on, when one 
is experimenting with single substances and no stain, 
the arrangement of the jellies is more simple, and the 
equation is not used so much. 

9 



CHAPTER VIII 

THE EXCITATION OF AMCEBOID MOVEMENTS IN WHITE 
BLOOD-CORPUSCLES CAUSED BY ALKALOIDS 

Soon after this in-vitro method of staining was invented, 
it occurred to me that it might be employed for 
measuring the lives of human leucocytes after their 
removal from the body. Much work had been done 
by others in the way of determining the effects of 
virulent disease-germs on men and animals, and soon 
after the discovery of "opsonins" by Wright and 
Douglas, many researches were made to find out how 
individual human cells defended the body by attacking 
pathogenic organisms; but little was known about 
the effects of virulent germs and their poisons on the 
protecting leucocytes. Hence, if one could measure 
the lives of leucocytes, it would be a simple matter 
to mix them with the toxins produced by bacteria, 
and determine the virulence of these toxins by seeing 
how long it took for them to kill leucocytes. 

In order to measure the length of time that 
leucocytes will live in a given sample of blood removed 
from the body, it is obvious that the first thing to 

130 



LIVING AND DEAD CELLS 131 

be done is to be able to distinguish accurately between 
a living and a dead leucocyte; it is impossible to say 
how long a cell will live if there is no means of telling 
when it is dead. It may appear strange, but it is a 
fact, that it took two years to find out the difference 
in the appearance between a living and a dead leucocyte. 
During this two years many of the points regarding 
the diffusion of substances into cells, vaeuolation, and 
achromasia were found out; but although many efforts 
were made experimentally to try to perfect a method 
of measuring the lives of leucocytes, this difficulty, that 
one could not accurately distinguish between living and 
dead cells, always stood in the way. When the point 
was discovered, it may almost be said that it was by 
accident, and even then its value as a method of 
measuring the lives of the cells was not appreciated 
for some time. 

It was known, of course, that leucocytes lived for 
some hours after their removal from the circulation, for 
they sometimes showed amoeboid movements; but 
in order to measure the lengths of their lives it was 
necessary to be able to say at any given moment that 
so many leucocytes in a given sample of blood were 
alive, and that so many were dead. The cells were 
always examined on jelly which contained stain; some- 
times they showed movements and sometimes they did 
not; but the absence of movements was no evidence 
that death had taken place. Many experiments were 
made, and at last it was resolved deliberately to kill 
some cells by a virulent poison, and to see whether the 
cells so killed appeared in any way different from those 



132 THE EXCITATION OF AMOEBOID MOVEMENTS 

not so treated. The poison was mixed up with the 
stained jelly, and that jelly was alkaline, in order to 
cause the diffusion of the stain and of the poison into 
the cells. The poison chosen in the first instance was 
hydrocyanic acid, and then nitrobenzol was used; but 
after subjection to them, the cells presented very little 
difference from others not so treated and known to be 
alive. At last atropine sulphate was tried, with a very 
astonishing and unexpected result, for every leucocyte, 
far from being killed outright, became excited to great 
activity. Some time afterwards it was realised that 
this excitation by atropine was very constant, and that 
if a cell was placed on a suitable jelly which contained 
atropine, it would, if alive, respond with absolute 
certainty by exhibiting excited amoeboid movements. 
Thus the means of measuring the lives of leucocytes 
was determined, and it became a simple matter, by 
examining the leucocytes in a given sample of blood — 
over a series of intervals — to discover how long they 
lived under varying conditions, for one was enabled 
at once to say whether the leucocytes were living or 
dead, the living ones showing exaggerated movements, 
the dead ones remaining immobile. 

This method of measuring the lives of leucocytes, 
and the details connected with it, will be found in the 
Appendix. It was originally intended to use it for 
ascertaining the effect of toxins on leucocytes, and we 
think that for this it will have a useful application. 
Owing to the fact that the excitation by the alkaloid 
led to other work, we have not yet had time to in- 
vestigate the actions of toxins very far. 



KINETIC JELLY 133 

Apart, however, from being able to measure the lives 
of leucocytes, it is very necessary in this in-vitro work 
to be able to tell at once when the cells with which 
we are experimenting are alive, for it is essential that 
one should deal only with living cells; hence we will 
now give the formula for the preparation of a suitable 
jelly which will excite amoeboid movements in living 
leucocytes. This jelly has been called for convenience 
"kinetic jelly," for it will always excite living leucocytes 
to activity. It is as well always to have a tube of it 
ready to hand, in order that at any time a film may 
be prepared, so that we may be able to make certain 
that the cells in a sample of blood are alive. It is 
prepared as follows: To a tube of 5 cc. of "coefficient 
jelly" add five units (0.5 cc.) of Unna's stain, six units 
of alkali solution (0 . 6 cc. of 5-per-cent sodium bicar- 
bonate), and 0.7 cc. of a 1-per-cent solution of atropine 
sulphate. The content of the tube is made up to the 
total of 10 cc, with 3.3 cc. of water. The mixture 
should be melted and boiled until it froths up in the 
tube, and a drop of the stained jelly poured on to a 
slide and allowed to set. A drop of fresh citrated 
blood is then placed on a cover-glass, which is inverted 
on to the film in the usual manner. It should be 
examined at the room temperature, which may be said 
to be about 18 or 20° C, 

When the cells come to rest on the jelly they will, 
of course, be unstained. Slowly their granules begin 
to turn red. A field which contains a few leucocytes 
should be watched. In about fifteen minutes it will 
suddenly be noticed that around the circumference of 



134 THE EXCITATION OF AMCEBOID MOVEMENTS 

first one leucocyte and then in the others small bodies 
like minute beads appear. These beads seem to come 
from underneath the cell. The beads get larger, and 
quickly develop into long snake-like processes of cyto- 
plasm, which are extruded from the cell. In a few 
moments every leucocyte in the specimen will appa- 
rently be putting out these long " feelers" until the 
cells may almost be said to look like tarantulas (fig. 28) . 
There are usually two or three of these long pseudo- 
podia extruded from each cell. At first the pseudo- 
podia are composed of clear cytoplasm (fig. 29), but 
later on a few granules from the cell are seen to move 
into them. Leucocytes seem to endeavour to push 
their pseudopodia into the crevices between the neigh- 
bouring red cells if they can (fig. 30), although we 
have no reason to give for this propensity. These 
excited movements differ from ordinary amoeboid 
movements in that they are far more exaggerated. 
The picture of a field containing several excited 
leucocytes is a striking one, for these movements are 
very different from the ordinary sluggish amoeboid 
movements seen when the cells are merely kept on 
a warm stage. Moreover, it must be remembered 
that we are using the room temperature and no warm 
stage. 

The excited movements are due to the action of the 
atropine. All the time, however, the stain is diffusing 
into the cells as well as the alkaloid, and as time pro- 
gresses the stain will reach the nuclei which now begin 
to turn a faint blue colour. Now, it has already been 
pointed out that the staining of the nucleus of a cell 



KINETIC JELLY 



135 




Fig. 28. — Amoeboid movements excited in a leucocyte by the action of 
atropine. Low power. - 






Fig. 29. — Exaggerated amoeboid movements in leucocytes which have their 
granules stained. The movements were excited by atropine sulphate. 



KINETIC JELLY 



137 






Fig. 30. — Excited leucocytes extruding their pseudopodia between red cells. 




Fig. 31. — Excitation of amoeboid movements in a lymphocyte by the action 
of atropine. No stain. 



KINETIC JELLY 139 

will kill it, and therefore all the leucocytes in the 
specimen slowly begin to retract their pseudopodia, 
for leucocytes endeavour to resume their spherical shape 
before death. The long snake-like processes can be 
seen to shrink back gradually into the cells (figs. 7,8), 
until in most cases they are completely retracted 
(fig. 9). Occasionally, however, a constriction appears 
in a pseudopodium where it arises from the cell-wall 
(fig. 26), and separation has actually been seen to take 
place; the separated portion, often containing a few 
cell-granules, will now resemble a blood-platelet. Soon 
after the pseudopodia have been retracted the cell dies, 
and either bursts or becomes achromatic. 

If the jelly has been properly prepared the whole 
phenomenon of excitation of amoeboid movements will 
be over in about twenty-five minutes or so. The action 
of this kinetic jelly is instructive, for it affords 
another example of the diffusion of substances into the 
cells, and of the accuracy of the equation used in its 
preparation in relation to this diffusion. The way of 
making the jelly has been described, and it must be 
remembered that it contains 0.7 cc. of a 1-per-cent 
solution of atropine sulphate. Now, this is a salt, and 
it delays diffusion; hence its unit must be ascertained 
before the correct equation can be made for this jelly. 
The unit of atropine sulphate (as found by experiment) 
is .013 gramme, and therefore since the jelly contains 
0.7 cc. of a 1-per-cent solution, it must contain . 5 of 
1 unit, which may now be added to the equation among 
the other salts which are minus factors. We may now 



140 THE EXCITATION OF AMOEBOID MOVEMENTS 

state the formula for the index of diffusion of this 

fx = (5s + 6a)-(3c+n + 0.5z), 

where z = the unit of atropine sulphate. 

The specimen is kept at the room temperature, or 
3 units of heat; and the object of the jelly is to excite 
amoeboid movements in fifteen minutes (or 1 . 5 unit 
of time) in neutrophile polynuclear leucocytes, which 
have a cf of 12. This jelly, of course, is arranged for 
the coefficient of diffusion of leucocytes, and it may thus 
be set down : 

cf = (5s + 6a + 3h + 1.5t)-(3c + n + 0.5z)=ll. 

Now, if these equations are carefully considered, it 
should be noticed that they are apparently wrong: the 
coefficient of diffusion of neutrophile leucocytes is 12, 
not 11. 

This brings us to another rule. It is obvious that 
if the jelly was prepared for the exact coefficient of 
diffusion of leucocytes, we would not obtain excitation 
of those cells in the given time — we would only obtain 
staining of their nuclei, and staining of the nuclei means 
that the cells would be dead. This would mean that 
we should defeat our object, for dead cells with their 
nuclei stained will certainly not respond to the atro- 
pine. ' 6 The determination of the coefficient of diffusion 
of nucleated cells involves death," because the stain- 
ing of the nucleus is the moment by which the cf is 
obtained. 

But this difficulty can be overcome by subtracting 



KINETIC JELLY 14-1 

one digit from the coefficient of diffusion, and making 
the jelly accordingly. Hence the equation given above 
in reality is correct, for the coefficient of diffusion of 
leucocytes is 12, and subtracting one digit from it makes 
11, as given in the equation. With the jelly arranged 
for 11, the nuclei are not yet stained, and death will not 
occur for another unit of time. On the other hand, the 
diffusion has already been sufficient for the atropine to 
excite the cells, and when the given fifteen minutes of 
time has elapsed, the cells will be seen, not dead, but 
in the height of their excitation. 

Thus the rule is that, having ascertained the co- 
efficient of diffusion of a cell, if we wish that cell 
to be alive at the expiry of the given time, subtract 
one digit from its coefficient of diffusion, and make 
the jelly accordingly. 

This rule is an important one in this work, for 
we shall, of course, frequently have to observe cells 
in the act of excitation, which is an easy matter if 
their coefficient of diffusion is known, as it only 
remains to subtract one unit from any of the factors 
which increase diffusion, and we get the right result. 1 

All forms of the polynuclear leucocyte respond 
to atropine by exhibiting excitation of amoeboid 
movements. In making them respond, however, the 
different coefficients of diffusion of each class of cell 
must be duly regarded. The eosinophile cell has a 
coefficient lower by one unit than the neutrophile; 
and if it is required to excite it especially, the jelly - 

1 It will doubtless be realised that subtracting one unit of a factor which 
increases diffusion, is similar in effect to subtracting one digit from the cf. 



142 THE EXCITATION OF AMCEBOID MOVEMENTS 

film must also have an index lower by one unit than 
that for the neutrophile corpuscle. The lymphocyte, 
or mononuclear corpuscle, also becomes excited to a 
marked degree by absorption of atropine (figs. 31-3) ; 
indeed they extrude longer pseudopodia than any of 
the other classes of blood-cells, a fact which is more 
interesting, because it is generally supposed that the 
lymphocyte is not a very amoeboid cell, a supposition 
which is erroneous. To induce amoeboid movements 
in the mononuclear cells, however, it is best to treat 
them as though they had a coefficient of diffusion 
lower than that of the leucocytes by about one unit, 
as these cells seem to die before the nucleus becomes 
stained. It was pointed out in the original specifi- 
cation that the staining of the nucleus indicated the 
point at which the coefficient of diffusion is determined. 
It has already been mentioned that this term nu- 
cleus is rather vague, and, as will be shown later, 
death is occasioned in the lymphocyte by staining 
of the nucleolus, which frequently becomes coloured 
before the nuclear wall. For general purposes, 
however, the original specification stands good. 

The foregoing experiment, by which one can 
excite amoeboid movements in leucocytes which have 
their granules stained, proves that the staining of 
their Altmann's granules is not very harmful to cells. 
The granules can, and do, become deeply stained, 
and all the while the cells will continue to extrude 
and retract pseudopodia in response to the alkaloid. 
This point is a very important one when we come 
to study induced cell-division, for it affords a clue as 






DIFFUSION OF TWO AGENTS 



143 




Fig. 32. — Excitation of amoeboid movements in a lymphocyte which has 
its granules stained. 




Fig. 33. — Extreme excitation of amoeboid movements in a lymphocyte. 

No stain. 



DIFFUSION OF TWO AGENTS 145 

to how the chemical exciters of reproduction act in 
the causation of mitosis. 

Another point is learnt, however, by experimenta- 
tion with this combination of stain and atropine, for 
here we have two chemical agents, an anilin dye 
and an alkaloid, both diffusing into the cells side by 
side and each producing its effect on the cell-proto- 
plasm. One excites the cell, the other kills it, and 
each carries out its function in direct proportion to 
its own concentration; for if the content of the stain 
in the jelly is reduced, the cells become less stained, 
and death is delayed; but if the alkaloid alone is 
reduced, the staining is as usual, but there is less 
excitation. At the same time, it must be remembered 
that the alkaloid is a salt, and, like other salts, as it 
diffuses itself into the cell, it delays the diffusion of 
the stain. 

The diffusion of a combination of substances into a 
cell, therefore, is not a simple matter, for it represents 
an equation of variables, although those variables, if 
applied in the same manner, always have the same 
effect with mathematical precision. 

The excitation of amoeboid movements in white 
corpuscles is due entirely to the atropine. Using a 
jelly-film which is alkaline, 1 and which contains stain 
but no atropine, no amoeboid movements will occur, 
and the cells retain their spherical shape. If the jelly 
is neutral, however, occasionally sluggish movements 
occur, even at the room temperature. At a tempera- 
ture of 30 to 37° C. sluggish movements may occur 

1 The alkalinity of these jellies is not sufficient to precipitate the alkaloids. 



146 THE EXCITATION OF AMCEBOID MOVEMENTS 

even in the presence of alkali. But in any of these 
instances the movements are not comparable to the 
deliberate extrusions caused by atropine, which are very 
striking in character, and if once seen will always be 
remembered. 

We can, of course, make kinetic jelly suitable for 
the temperature of the blood (it is merely necessary 
to reduce the content of alkali in the jelly by 3 units, 
because we increase the temperature by 3 units), and 
still the excitation occurs, although (and this is a 
remarkable circumstance) the excited movements are 
not so marked at the temperature of the blood as they are 
at that of the room. Many persons who have seen the 
action of kinetic jelly have disparaged it, saying that 
they have often seen marked amoeboid movements in 
leucocytes; but when questioned, the fact is always 
elicited that they have employed the warm stage. It 
is the deliberate and constant exaggerated movements 
which invariably occur in all living leucocytes at low 
temperatures which constitutes the striking effect of 
atropine sulphate upon them. Let a control experi- 
ment be made with a jelly which contains no atropine — 
and no stain either if one wishes to — and the difference 
is immediately apparent. Excited by the alkaloid, the 
cells with their stained granules, extruding their long, 
snake-like pseudopodia in all directions, as if they were 
searching for something (which, as far as can be found 
out, they are not), form a very pretty picture, which, 
when seen through the microscope, will be a revelation 
to those who have only worked with films of dead cells. 

Atropine sulphate is not the only substance which 



NOT DEATH-STRUGGLES 147 

causes this excitation. We have tried several alkaloids, 
and all of them have had this effect . It does not matter 
what the alkaloid is, nor whether it is a salt or an 
alkaloid; the result is the same. In fact, we think 
that it is probable that this power of exciting amoeboid 
movements is a property of alkaloids generally. It is 
true that we have not yet tried all known alkaloids, 
but we have experimented with many, and we think 
that they probably all have this effect. Moreover, the 
parent substances of alkaloids, such as pyridine and 
quinoline, also excite white blood-corpuscles. 

Some alkaloids cause more excitation than others; 
atropine has so far proved the most effectual, morphine 
the least. To man atropine is very poisonous; mor- 
phine is not so poisonous, weight for weight. To a 
man's leucocyte, however, it is curious to note that 
morphine is the more poisonous, and atropine not 
nearly so dangerous. By means of this jelly method 
we can try the effects of alkaloids and substances in 
various strengths on leucocytes and other cells, and if 
the jelly contains atropine, by noting the extent of the 
excitation one can find out the dose of an alkaloid 
which will cause maximum excitation and the dose 
which will cause death in a given time. Generally 
speaking, it requires three times as much of a given 
alkaloid to cause death as it does for it to cause 
maximum excitation. 

This latter point is an important one, for it has 
been suggested to us that the excitation by alkaloids 
is in the nature of a death-struggle. It is clear, 
however, that if it was, the excitation would steadilv 



148 THE EXCITATION OF AMCEBOID MOVEMENTS 

increase as more alkaloid was absorbed; but such is 
not the case. Moreover, this excitation is not caused 
by poisons, such as nitrobenzol and prussic acid. The 
possibility of the excitation being due to a death- 
struggle is also precluded by the fact that if no 
stain is employed the excited movements may be 
watched for an hour. Death-struggles, as seen in 
higher animals, do not usually last very long, and 
always commence immediately before dissolution. The 
excitation appears to be a specific one caused by 
alkaloids, although we have seen a similar form of 
excited movements, but not to the same extent, 
caused by arsenic. 1 

As already mentioned, we have ascertained the 
amounts of other alkaloids which cause maximum 
excitation of leucocytes, and in finding out these 
"doses" we have always used a similar jelly containing 
no stain, and the temperature employed has been that 
of the room in every case. The jelly was alkaline, as 
it contained 5 units of alkali solution, and the alkaloids 
were each used in a 1-per-cent solution, thus: To 
5 cc. of coefficient jelly, 5 units of alkali solution, and 
the amount — whatever it is — of alkaloid solution were 
added, and the balance, up to the usual total of 10 cc, 
was made with water. The jelly was then boiled, and 
a film prepared from it in the usual way, fresh citrated 
blood being used in each case. 

The following is a list of alkaloids which we have 
tried on leucocytes, and the amount of each of them 

1 The effects of oxygen have been tried on leucocytes by bubbling the gas 
through the jelly; but its action seemed to be negative. 



STRENGTHS OF ALKALOIDS 149 

which, when mixed with the jelly, produces maximum 
excitation. Treble this amount, and death will gener- 
ally occur without excitation, although leucocytes will 
stand even ten times the dose of codeine and bruceine 
without dying. 

To produce maximum excitation in twenty minutes: 

Alkaloid. Amount of 1-per-cent solution of it 

contained in 10 cc. of jelly. 

Bruceine 1 cc. 

Morphine 0.2 

Pilocarpine Nitrate 0.5 

Cocaine Hydrochloride .... 2 

Strychnine 1 

Atrophine Sulphate 0.7 

Aconitine 0.5 

Codeine 3 

Atropine is undoubtedly the most active of the 
vegetable alkaloids; but, as will be shown later 
choline (figs. 34, 35), and cadaverine (fig. 36), two 
of the animal alkaloids produced by putrefaction, are 
nearly as effective. The action of morphine in exciting 
exaggerated movements is very poor (fig. 37), but still 
it does have this effect. The dose may be doubled 
with cocaine, and the excited movements continue. 
Strychnine is not so effective an excitor for leucocytes 
as atropine. Codeine acts more effectively than 
aconitine. Pyridine is fairly effective (fig. 28) . 

The excitation of leucocytes by alkaloids is a very 
remarkable thing, for it seems to be a functionless 
procedure on the part of the cells. The alkaloids do 
not appear to cause the cells to migrate at all; they 
remain in their original position, and merely extrude 



150 THE EXCITATION OF AMCEBOID MOVEMENTS 

and retract their pseudopodia aimlessly. Quinine 
hydrochloride excites thern fairly markedly; and it 
must be noted that the statement has been made by 
other authors that quinine stops diapedesis. We have 
made a hanging drop preparation 1 with a jelly-film 
in such a way that the cells are not pinned down by 
the cover-glass, but still absorb atropine, and they 
therefore were in a position to move about if they 
wished to. They remained in their original positions, 
however, and seemed to be content to extrude and 
retract their "feelers." 

Experiments were made to see if this excitation 
was due to any chemotactic influence of the alkaloids. 
Two jellies were made, one of which contained atropine 
and the other none; and they were so mixed on a 
slide that there was atropine in one part of the film 
and not in another. Some citrated blood was placed 
over the line of demarcation to see if the cells neces- 
sarily extruded their pseudopodia in the direction of 
the concentrated alkaloid. They did not do so, how- 
ever, for, provided a cell absorbed the alkaloid suffi- 
ciently, the extrusions were made in all directions as 
usual. 

In order to try to find out whether this excitation 
increased the ingestion of bacteria by leucocytes, a 
sample of fresh blood was mixed with a volume of 
citrate solution and atropine, which contained bacteria 
in suspension. Having been incubated for some 
minutes, the cells were spread on jelly; but when 

1 This method will be found in the Appendix, where also another method 
of preparing kinetic jelly will be found. 



EXCITATION AND PHAGOCYTOSIS 151 




Fig. 34. — Excitation of two leucocytes by the action of choline. Low power. 

No stain. 




Fig. 35. — Excitation of a lymphocyte by the action of choline. No stain. 



EXCITATION AND PHAGOCYTOSIS 



153 




Fig. 36. — Excitation of amoeboid movements in a leucocyte by the action 
of cadaverine. No stain. 



Fig. 37. — A leucocyte excited by morphine. The cell's granules are stained. 



EXCITATION AND PHAGOCYTOSIS 155 

the number of bacteria ingested were compared with 
those phagocytosed in control experiments where no 
alkaloid was used, it was seen that the excited cells 
did not ingest more germs than usual. Excitation, 
therefore, does not increase phagocytosis; and we have 
noticed that if a mixture of living leucocytes and 
germs are mixed and spread on jelly which contains 
atropine, the cells do not purposely extrude their 
pseudopodia in the direction of any bacteria which 
may be near them. On the contrary, if a pseudo- 
podium happens to strike against a bacterium, the 
latter is usually pushed out of the way. 

Whether leucocytes are excited or not, we have 
never seen a cell actually ingest bacteria. We have 
often seen cells with bacteria inside them, but we 
have never seen the actual act of ingestion, nor have 
we any explanation to offer as to how it occurs. 
Moreover, we have often seen leucocytes with red 
cells apparently inside them, although how they came 
to be absorbed we do not know. It is possible that 
the laws of diffusion may play some part in the 
actual act of phagocytosis. 

iVnother point in connection with phagocytosis may 
be mentioned. In the making up of fixed films, 
germs and other substances may be crushed into 
leucocytes. By the examination of living cells this 
cannot happen. We have seen fixed specimens which 
showed phagocytes apparently crammed with germs; 
but on looking at another sample of the same cells 
alive a very different impression was obtained. We 
have mentioned this point in view of the possibility 



156 THE EXCITATION OF AMCEBOID MOVEMENTS 

of fallacy arising in the technique of the "opsonic 
index," if it is carelessly carried out, because in that 
technique fixed films are usually employed. 

The possibility of foreign substances being crushed 
into cells during the preparation of fixed films is also 
the reason, we think, for the common, fallacious 
supposition — which has already been mentioned — that 
the blood-platelets are the extruded nuclei of red 
cells, for in the preparation of fixed films platelets are 
crushed into red cells, to which they often adhere, and 
after fixation they appear as if' they were emerging 
from them; an artefact never seen with the jelly 
method. 

In concluding this chapter it should be mentioned 
that Professor Osier, many years ago, pointed out 
that certain alkaloids excited amoeboid movements 
in leucocytes, although this fact was not known to 
me when the effects of atropine mixed with the jelly- 
film were first tried. 

As will be shown later, alkaloids have a far more 
important action on cells than merely exciting amoe- 
boid movements, for they greatly augment the action 
of the exciters of reproduction. 



CHAPTER IX 

the adoption of the in-vitro method for cancer 

research the excitation of leucocytes 

caused by cancer plasma facts known 

about cancer the age incidence; vitality; 

death; metastases; chronic irritation — the 
possible causes of cell-proliferation dis- 
CUSSED. 

In August, 1908, on an occasion when the excitation 
of leucocytes by atropine was being demonstrated to 
one of us (C. J. M.) he remarked that he had often 
enough thought that patients dying from cancer ex- 
hibited symptoms resembling those of poisoning by 
some alkaloids, and he suggested that an investigation 
might be made by means of the in-vitro method to 
try to find out whether there existed in the blood 
of cancer patients any substance of an alkaloidal 
character which might be responsible for these 
symptoms. This suggestion, based on bed-side obser- 
vation, taken in conjunction with the fact that a group 
of chemical agents existed which were capable of 
exciting human cells, warranted a research in this 
direction, for if such a substance existed in the blood 
of these patients it was felt that either it might have 

157 



158 APPLICATION TO CANCER RESEARCH 

some bearing upon the cause of the disease or that 
it might be an effect of it. 

A cancer consists essentially of cells of the body 
which have multiplied irregularly and too rapidly, and 
it was quite reasonable to think that this form of 
excessive proliferation might be the result of some 
abnormal excitation. 

It should be remembered that in August, 1908, 
the actual cause of cell-division was quite unknown, 
and multiplication of individual human cells in direct 
response to a chemical agent had, of course, never 
been seen. It was realized that the problem of the 
nature of cancerous growths could only be solved by 
the discovery of the cause of cell-division. The cells 
of the body are continually multiplying by cell-division, 
and the correct appreciation, not only of the nature of 
new growths, but also of the problem of healing, and 
in reality most of the problems of pathology, depend 
upon the cause of cell-reproduction. 

The most commonly accepted theory regarding the 
cause of cell-proliferation was that cells divided owing 
to some inherent vital propensity — that is to say, that 
they multiplied because it was their "duty" to do so. 
As a matter of fact, however, nothing was known as 
to the immediate cause of individual cell-reproduction. 

So much work had been done with reference to 
cancer, and in spite of it so little was known concerning 
the cause of that disease, that we felt justified in follow- 
ing any clue, however slender it might appear at first 
sight. It was true that the excitation by alkaloids had 
so far only resulted in the production of exaggerated 



EXCITANT IN CANCER PLASMA 159 

movements in white blood-cells; but still it was an 
excitation, and for all we know, although they had not 
yet been seen, the excitation might produce other 
results as well. This clue, however, arising from micro- 
scopical experimentation and from a clinical observation, 
has proved to be of great importance, and has led by a 
singular chain of events to the knowledge that cell- 
division in the body results from the presence of specific 
agents, the action of which becomes remarkably aug- 
mented if the cells are in a condition of excitation 
resulting from the presence of an alkaloid. 

This cancer research became instituted in this way, 
and the first step undertaken in connection with it 
was to test the blood of cancer patients experimentally 
in order to find out whether it, or other of the body 
fluids, contained any substance which would, like the 
alkaloid, excite exaggerated movements in leucocytes. 

Ten cases of well-marked carcinoma were examined 
in the following way: A certain quantity of the 
patient's blood was mixed in a capillary tube with 
an equal volume of citrate solution. The tube was 
then centrifugalised and the corpuscles removed. To 
the remaining plasma a certain quantity of fresh blood 
taken from a healthy person (usually one of ourselves) 
was added and thoroughly mixed. The sealed tube 
containing the mixture was then placed in the revolving 
apparatus in the incubator and kept at 37° C. for half 
an hour, at the end of which time a drop of it was 
examined at 20° C. upon a slide under a cover-glass. 
Blood plasmata taken from fifty healthy people, or from 
people suffering from diseases other than cancer, were 



160 APPLICATION TO CANCER RESEARCH 

similarly tested, and it was found that the leucocytes 
of healthy people bathed in the plasmata of cancer 
patients undoubtedly showed amoeboid movements 
which were exaggerated and different in character from 
those which were observed in the corpuscles suspended 
in the plasmata of normal persons, or of persons 
suffering from a number of other diseases. The differ- 
ence was one of degree, however, for leucocytes fre- 
quently under these conditions showed some amoeboid 
movements; but we were quite satisfied that there was 
a distinct difference, although the test could not be 
considered a very delicate one. 1 

This series of experiments made us consider that 
there probably is some agent in the body fluids 
of cancer patients which causes excitation of cells, 
and one of us was charged with the task of further 
confirming the correctness of this observation, and 
of finding out what the substance is and how it is 
produced. It was appreciated that this substance 
could only be present in the blood in small concen- 
tration, and that to isolate it from serum would prove a 
very difficult task. 

As a preliminary to this part of the research, it 
was considered advisable to review the known facts 
concerning cancer to see whether they harmonised 
with the possibility of the disease being associated 
with an excitation of cells by chemical agents. After- 
wards we proceeded, by means of the new jelly method, 
to try the effects of different substances either taken 

1 A paper by Dr. Macalister and myself describing these experiments was 
read before the Royal Society of Medicine in November, 1908. 



AGE-INCIDENCE 161 

from growths, or which we knew were associated 
with growths, on individual living healthy cells. By 
this means it was hoped that we might find some 
exciting substance from cancerous growths which 
might in the first place cause normal individual cells 
to undergo a change and become similar to those 
cells taken from the growths themselves. In the 
event of such a substance being found, it would, of 
course, then be necessary to try to prove the argu- 
ment by experimentation with the substance in the 
body itself. In other words: believing that cancer 
might be due to a chemical agent, we proposed to try 
to find that agent, and to test its effect, in the first 
instance on individual cells under the microscope, and 
lastly to test its action on groups of cells in the tissues 
of the body. 

Malignant disease may be separated into two 
main divisions — carcinoma and sarcoma. The former 
attacks gland-tissues and epithelial cells; the latter is 
a disease of connective tissues. These researches are 
almost entirely concerned with carcinoma, and the 
term "cancer" in this book refers .to that disease. 
There is, we think, a close association between these 
two forms of malignant disease, although there is 
a line of demarcation in the age incidence and in 
some of their morphological and clinical characteristics 
which separates them. Cancer — that is to say, 
carcinoma — attacks people over the age of forty, 
although there are occasional exceptions to this rule; 
but sarcoma may occur at any age from infancy onwards. 
At the outset we turned our attention exclusively to the 



162 APPLICATION TO CANCER RESEARCH 

consideration of carcinoma, for we considered that if we 
succeeded in throwing any light on the causation of that 
disease, it would be time enough for us to investigate 
sarcoma. Cancer is much more common than sarcoma ; 
but it has to be remembered throughout that, from 
the similarity of the cardinal symptoms of the two 
diseases, there is probably an intimate association 
between the causes of both. The connective tissues 
can become malignant at any age. The epithelial 
tissues are usually attacked after the age of forty. 
This age-incidence of carcinoma is most striking, and 
it necessarily constitutes a fundamental fact with 
which all our thoughts regarding the cause of 
cancer must ultimately harmonize. It is a disease of 
senescence; it attacks people when they are robust and 
apparently in a state of highest vitality, just when they 
are in the prime of life, or having just passed it. We 
have to remember in this connection that the expres- 
sion "prime of life" in its physiological sense may be 
taken to refer to middle life — that is, somewhere about 
the age of 35; and we may further understand that 
before that age a man is being built up, whereas after- 
wards he enters upon the downward trend and steadily 
progresses towards physiological death, which may be 
taken to occur about the seventieth year. We may 
therefore consider that the climax of his physiological 
life is reached at 35. 

The age incidence of cancer is unique; there is no 
other disease which has this limitation in its age 
averages. Exceptions do occur, it is true, but the 
number of cases occurring during senescence, when 



VITALITY 163 

man has passed the climax of his age, is so enormous 
that the possibility of fallacy due to "the error of 
random sampling" is reduced almost to zero. It is a 
salient feature of the disease which cannot be disputed, 
and we may regard is as an axiom that cancer attacks 
people when they are trending downwards from their 
physiological prime. The question is, therefore, What 
happens in the tissues during this senescence which 
renders them liable to the onset of cancer ? At the 
time when these researches were first applied to the 
investigation of cancer, this question could only be 
answered in a speculative manner; but it was appre- 
ciated that the conditions present after the prime of 
life which predisposed to the disease might merely 
depend on something in the nature of the oversetting 
of a physiological balance. 

Vitality seems to be worthy of consideration as a 
factor connected with the onset of malignancy. Verv 
old persons do not appear to be so liable to cancer 
as those between the ages of 40 and 55 — a circum- 
stance which may possibly be due to a loss of vitality, 
for it has already been mentioned that cancer is a 
disease of the robust. Premature ageing, on the other 
hand, seems to favour the onset of cancer; but in 
conditions of decrepitude there is more freedom from it. 
Tottering persons, such as are seen in asylums and 
institutions, do not so frequently develop carcinoma; 
but people who are sufferers during their senescence 
from the atrophic form of osteo-arthritis or from gout 
are common victims. Let the reader visit a home for 
incurables, and he will there learn that manv of the 



164 APPLICATION TO CANCER RESEARCH 

cases of cancer arising in the institution are also afflicted 
with rheumatoid arthritis. The aetiology of cancer is 
a large subject, and for full information regarding 
what is known of it reference may be made to an 
excellent volume by Mr. W. T. Gibson, on The 
Etiology and Nature of Cancerous and other Growths. 

This book enumerates in detail the trades and pro- 
fessions the members of which are especially prone to 
cancer, and it furnishes a valuable aid to pathological 
cancer research. Therein it is shown that chronic 
alcoholism is a predisposing factor. Syphilis also is 
undoubtedly a predisposing cause of cancer, provided 
the disease is not too severe. We have been reminded 
of this point by Mr. Pernet, 1 whose experience of 
syphilitic patients has left him convinced of an associa- 
tion between the two diseases. 

The conditions of decrepitude and chronic enfeeble- 
ment — to which reference has been made as ones which 
render persons less liable to malignancy — affect not 
only the general vitality of the body, but also pre- 
sumably the vitality of the individual cells. 

Cancer is a disease which is general throughout the 
world as far as we can find out, but climatic conditions 
appear to influence its incidence to some extent. Sir 
William MacGregor has told one of us that as far as 
he can remember he has never seen a case among the 
Esquimos, an observation which is interesting in con- 
nection with the association of cancer and some 
putrefactive products, which will be discussed in the 

1 "The Intramuscular Syphilitic Treatment/' by George Pernet, Transac- 
tions of the American Medical Association, June, 1909. 



CHRONIC IRRITATION 165 

later chapters of this book, for, generally speaking, 
putrefaction of organic substances must be reduced to 
a minimum in the ice-bound regions of the far North. 

Death is the ultimate result of cancer in most 
instances, unless the progress of the disease is success- 
fully interrupted by surgery, and this is a fact which 
must be carefully considered. Cancer consists of a 
growth, of human cells. Why should such a growth 
kill the person it afflicts. Benign growths do not 
necessarily cause death ? It may be, of course, that 
the original cause of the disease increases with the 
growth, and that it is this cause which is instrumental 
in killing the victim. We speak of death from cancer 
as resulting from the vague condisions described as 
"exhaustion and cachexia," but why these conditions 
result from cancer was not even within the realms of 
speculation. 

Cancer is a disease which seems to aggravate itself. 
Once the disease is started in the circumscribed area 
— for it always begins in one spot — it will go on steadily 
if it is left to itself. Moreover, one of the features of 
a malignant growth is that it produces metastases. Why 
should malignant growths and not benign ones produce 
metastases ? It is usually considered that metastases 
are due to embolism, and that the transplanted cells con- 
tinue to multiply in their new surroundings; but, again, 
why should these emboli only continue to mutiply in 
malignant tumours ? Benign growths, like the malignant 
ones, are supplied with vessels and lymphatics, and 
there seems to be just as much reason why portions of 
both forms of growth should be swept away to form 



166 APPLICATION TO CANCER RESEARCH 

metastases in other parts of the body. Still, the fact re- 
mains that metastases occur only in malignant disease. 

The foregoing points formed our axioms. Whatever 
experiments we undertook had to harmonise with them 
all in their consideration. There was one other factor, 
however, which has already been mentioned; the mys- 
tery of the cause of cell-division in the body, and a 
well-known predisposing factor in the causation of 
cancer which is intimately associated with it, namely, 
chronic irritation. 

The body consists almost entirely of living cells; 
individual living creatures, each of which is capable of 
separate existence for a short time, but which in con- 
junction with one another form the tissues which in 
their turn have special functions. Each cell is merely 
an individual in a multitude ; a unit in an organ. Cells 
not only have functions to perform for their own 
individual welfare, but they also act collectively for the 
general welfare of the body. 

Since cancer consists of a tumour composed of cells, 
we may attack the problem of its causation from two 
aspects — the investigation of the individual cells, and 
the investigation of collective masses of them. At the 
outset, the first aspect is obviously the one to receive 
consideration; and since cancer consists of a growth of 
cells which have multiplied too often and have so 
formed a tumour, the first question to be asked is, 
What makes this excessive multiplication take place ? 

Before this question can be approached, however, 
another question must be answered, namely, What 
makes any multiplication of cells take place ? 



WHAT MAKES CELLS DIVIDE ? 167 

The multitudes of cells which form our bodies have 
been evolved from a single pair of cells. The maternal 
ovum is a single cell, and always remains as such until 
it is fertilised by the paternal spermatozoon, which in 
its turn is also a single cell. The conjugation of the two 
at once causes cell-division to take place within the egg. 
Multiplication occurs, and where there was one cell 
there are now two; and each daughter cell divides and 
divides until generation after generation of new cells 
are produced, and this cell-reproduction ultimately 
leads to the formation of the new individual. The 
basis of the formation of new beings is the reproduction 
of cells by their division in response to the conjugation 
of an original pair of cells. We had therefore to ask 
ourselves why this conjugation should cause cell- 
division; but unfortunately the answer was unknown. 

Throughout our lives, although we cannot actually 
feel it, the cells in our bodies are continually repro- 
ducing themselves by division by mitosis, and individual 
cell-death is also constantly taking place. It is true 
that some cells, such as some cells of the nervous 
system, probably live throughout the length of our 
lives, but myriads of other cells are constantly dividing 
to help to build up the tissues. "Birth" and death 
are continually going on among the individual units 
of ourselves. When a tissue is sectioned and examined 
microscopically it will frequently be seen that some of 
the cells are in the act of division by mitosis; but when 
we asked what makes the division occur, and what 
makes cells multiply to build up the tissues, we could 
only say once more that the reason was quite unknown. 



168 APPLICATION TO CANCER RESEARCH 

When a child grows to form a man he grows by the 
multiplication of his cells, but we did not know what 
causes him to grow, or what makes his cells to attain 
this object. 

Again, if we injure or wound ourselves in any part 
of the body, the tissues always make an attempt to 
repair the damage. No matter to what extent the 
injury may occur, attempted healing always takes 
place. The phenomenon of healing is due to the 
proliferation of white blood-cells, which multiply by 
cell-division to repair the tissues which are damaged. 
Not only do leucocytes and lymphocytes proliferate 
when a tissue is damaged, but other cells also multiply. 
For instance, epithelial cells will also proliferate to heal 
a damaged site. The cell-proliferation of healing forms 
one of the bases of pathology, and therefore of medicine 
also; yet it had to be admitted that nothing whatever 
w T as known as to why this cell-proliferation occurs when 
any part of our bodies is damaged. The process of 
healing is occurring in our bodies throughout our lives, 
and this sudden multiplication of cells must be con- 
stantly before the consideration of medical men; but 
although this multiplication by reproduction is an 
established fact, one never hears the question asked, 
Why do cells immediately divide to reproduce them- 
selves when a tissue is damaged ? If the question was 
asked, however, the answer would have had to be, 
"We do not know." 

For the cell-proliferation of healing to occur it is 
not necessary for the skin to be actually broken. On 
the contrary, extensive cell-proliferation of healing may 



PROLIFERATION OF HEALIXG 169 

occur as the result of a bruise or disease; and chronic 
irritation, which is an invariable predisposing factor in 
cancer, may give rise to exuberant multiplication of 
the cells in the neighbourhood of the irritated part. 
A common instance of the proliferation due to chronic 
irritation is shown in the case of a "corn." An ill- 
fitting boot irritates a certain portion of the foot by 
pressing unduly on a certain portion of the skin. The 
skin becomes hardened, and a small tumour may even 
be formed on the irritated spot. This hardening is 
due to excessive proliferation of the living cells in 
and immediately underneath the skin. A wart is 
an example of the proliferation due to irritation; 
but although this irritation leads to proliferation, we 
do not know exactly why the cells proliferate in 
response to it. If we think the problem out care- 
fully, we can picture a group of living cells multiplying 
by division, and then try to grasp how irritation of 
that group by mechanical pressure can possibly make 
the individual cells reproduce themselves; for this is 
what they do. At the time when these cancer re- 
searches were started we could not grasp this point. 
It seemed incredible that a cell would reproduce itself 
because its cell- wall was "tickled" or pressed upon. 1 
Why should a living cell undergo the complex phe- 
nomenon of mitosis for a reason of this nature ? Be- 
sides, living cells are very delicate, and we know that 
they will not stand much handling or pressure without 
dying. No; it was necessary to find some better 
explanation of the cause of cell-division than mere 
mechanical irritation, and we appreciated that irritation 

ir The pressure of a cover-glass does not cause cell-division. 



170 APPLICATION TO CANCER RESEARCH 

in reality causes localized cell-death, and the cause 
of the proliferation due to irritation would in all 
probability be found to be due to the same cause or 
causes which make our cells multiply in order to heal 
up a cut or a sore. 

Now, there can be no doubt that cancer occurs in 
sites where there has been previously some form of 
chronic irritation, and cancer is another name for 
malignant proliferation of cells. Since such irritation 
is probably directly associated with the cause of the 
cell-proliferation of healing, we made our first endea- 
vours to try to find out this cause of cell-reproduc- 
tion in the body, for it was considered that if that 
could be found a step in the right direction would 
be made. Some attempts at healing are always going 
on in the parts that are subject to chronic irritation, 
and we may safely say that the cell-proliferation of 
healing is going on in these parts. Cancer super- 
venes on old ulcers and sores, which, of course, are 
healing sites. In the breast and uterus, two of the 
commonest places for cancer, the cell-proliferation of 
healing occurs every month during the ages of actual 
sexual function, and at the climacteric a large involu- 
tion takes place, accompanied by destruction of tissue. 
The irritation which causes cancer of the lip is usually 
the pressure of a tobacco-pipe; X-ray cancer usually 
follows the ulceration and damage due to burns; and 
there are many other examples. The cause of the 
cell-proliferation of healing, therefore, constituted our 
first investigation in the path of cancer research. 
Leucocytes and lymphocytes are the cells which pro- 



PROLIFERATION OF HEALING 171 

lifer ate to a great extent when healing occurs anywhere, 
and these white blood-corpuscles formed the objects 
of our observation in the first instance. What made 
these cells divide we did not know, how they divided 
was also unknown; but we knew that amoeboid move- 
ments could be excited in them by means of alkaloids. 
It is an astonishing thing that when any injury 
occurs, in no matter what part of the body, those 
neighbouring cells which have not been damaged will 
immediately reproduce themselves. If the damage is 
persistent, and healing becomes very chronic in persons 
over the age of 40, cells may reproduce themselves 
in a malignant manner, and then they go on dividing 
and dividing, producing a cancerous growth which 
ultimately kills the person the part of whose body 
they are, and whose damaged tissue it was their en- 
deavour to heal. The first thing to do, undoubtedly, 
was to try to find the cause of the cell- proliferation 
of healing. 



CHAPTER X 

EXPERIMENTS WITH NUCLEIN THE LOWERING OF THE 

COEFFICIENT OF DIFFUSION CAUSED BY EXTRACTS 

OF DEAD HAEMAL GLAND DIVISIONS INDUCED IN 

LYMPHOCYTES FOR THE FIRST TIME REVELATIONS 

CONCERNING THESE DIVISIONS THE ROLES PLAYED 

BY THE ALTMANN'S GRANULES, NUCLEI, AND 
NUCLEOLI IN THEIR CELL-DIVISION 

Before proceeding to discuss the problem of the 
causation of cell-division, it is necessary to state that 
another piece of information was at our disposal which 
we believed to be intimately associated with cell-division, 
although the fact was not appreciated when the point 
was first noticed. During experimentation with a 
mixture of stain and alkaloid on blood-cells it was 
noticed that with a citrated mixture of Unna's stain 
and the alkaloid atropine the lymphocytes sometimes 
extrude granules (fig. 39) from their cell-walls. These 
granules remain attached to the cell by means of a 
streamer, apparently derived from the cell-wall itself. 
The extrusion appears to be a deliberate one on the 
part of the cell, and the granule ultimately becomes 
separated from it altogether. This extrusion, or "flagel- 
lation" as we erroneously called it, has been confirmed 

172 



FLAGELLATION 



ITS 





Fig. 38. — Leucocytes excited by pyridine. No stain. 




Fig. 39. — A lymphocyte which has absorbed stain and atropine discarding 
its granules (flagellation) . 



6 ' FLAGELLATION " 1 75 

by L'Engle, of Philadelphia, who has also seen it occur 
in poly nuclear leucocytes. When we examined fresh 
blood-cells mixed with the plasma of cancer patients, 
we again noticed that the lymphocytes extruded 
granules in some cases apparently in response to 
something in the cancer plasma, a point which Dr. 
Macalister and I published in The British Medical 
Journal on January 16, 1909. Dr. Buchanan, how- 
ever, has informed us — and this is a most interesting 
point — that he had previously seen similar extrusions 
take place in cases of leukaemia, a fact which he 
mentions in his book; and a fact which we shall 
recall later on. We, however, had never seen these 
extrusions occur unless alkaloid or cancer plasma had 
been mixed with the cells. 

As already mentioned, the commonly accepted 
explanation regarding the cause of the reproduction of 
cells by individual cell-division is not very satisfactory. 
One of the characteristics of living matter is that it 
is capable of reproducing itself, and the theory as 
to its causation in animal cells was that they, being 
living creatures, reproduce themselves because it is an 
intrinsic function of the protoplasm — that is to say, that 
it is a vital propensity on the part of every cell to 
divide automatically, so to speak, and to continue to 
do so until it dies. This explanation, however, does 
not harmonise with certain known facts concerning cell- 
proliferation. For instance, physiologically cell-division 
is influenced by conditions outside the cell. The limi- 
tation of the size of an organ must be controlled by 
some governing factor which influences not only the 



176 DIVISIONS INDUCED IN LYMPHOCYTES 

proliferation of individual cells, but that of multitudes 
of them. It is very difficult to believe that the develop- 
ment of an animal from the ovum can be entirely an 
automatic function of the protoplasm of individual cells, 
unless that function is so controlled that the cells act 
together in masses. Moreover, the phenomenon of 
healing which has been mentioned presents features 
which tend to dispose of the "automatic theory" — a 
theory which does not explain why cells immediately 
reproduce themselves at a much quicker rate than 
normally when a tissue is damaged. Leucocytes, for 
instance, will not divide when they are removed from 
the body, nor have they ever been seen in the act of 
division when examined from the peripheral circulation. 
Yet when these cells are shed into a damaged tissue 
they proliferate immediately. 

Jacques Loeb was, we believe, the first to show that 
cell-division in the ova of star-fish can be accelerated 
by certain chemical reagents; and further observations 
were made in this line of work by B. Moore, H. E. Roaf, 
and E. Whitley, who proved that the regularity and 
rapidity of growth of the cells of the fertilised ova of 
echinoderms could be greatly influenced by certain 
alterations in the alkalinity of the water in which they 
normally divide. B. Moore has also shown that the 
alkalinity of the blood-plasma in cancer is increased — 
a point which is of great importance, especially when 
we remember that alkalies increase the diffusion of 
substances into living cells. 

O. and R. Hertwig and Galleoti have described 
how, when mitosis occurs in some of the cells of 



THEORIES OF CYTOGENY 177 

lower animals which have been subjected to certain 
alkaloids, such as quinine, nicotine, and cocaine, and 
also to antipyrene, the mitotic figures may be of the 
asymmetrical type, and that in the case of certain epi- 
thelial cells of salamanders the mitotic divisions which 
occur in the presence of these substances closely re- 
semble the asymmetrical divisions seen in human 
cancer-cells. These points took us back once more 
to our own knowledge that alkaloids excited leuco- 
cytes; but we have never seen divisions, asymmetrical 
or otherwise, actually induced in leucocytes by any 
alkaloid or other substance. 

Farmer, Moore, and Walker had closely studied 
the cytology of cancer-cells. They had frequently 
seen cells in the act of division in their stained speci- 
mens, and they described the asymmetrical "maiotic" 
mitoses by which cancer-cells frequently appear to 
divide. By the expression "maiotic division" a "re- 
duced" division is meant; that is to say, that a cell 
divides with a reduced number of chromosomes, and 
instead of having its customary somatic number, that 
number may be reduced to one-half. In man the 
somatic number of chromosomes is thirty-two, and 
cancer-cells sometimes divide with sixteen chromo- 
somes. Farmer, Moore, and Walker also describe 
other characteristics of the several maiotic phases of 
mitosis, and they specify two forms of maiotic di- 
vision — namely, the first change in a cell's life- 
history from its somatic division to the maiotic, which 
they call the first (heterotype) maiotic division, and the 
succeeding maiotic divisions of its life-history, which 



178 DIVISIONS INDUCED IN LYMPHOCYTES 

are called the homotype maiotic divisions. These 
authors, however, believe that it is not only cancer- 
cells which divide by maiotic divisions, but that certain 
other tissue-cells also normally proliferate by maiotic 
reproduction, especially some cells of the testis, and 
the "wandering" cells of the body. 

In March, 1909 we discussed the problem of the 
causation of cell-division with Professor Harvey Gibson, 
who suggested that we might try the effect of nuclein 
on cells; and he founded this idea on the well-known 
fact that in the sexual generation of the normal alter- 
nations of generations of plants the nuclei have only 
half the number of chromosomes which are present in 
the nuclei of the asexual generation, and that what is 
normal in the plant appears to resemble what is patho- 
logical in the human being's cancer-cells. It is thus 
suggestive that a cancerous growth might be looked 
upon as consisting of abnormally induced "gameto- 
phytic" or sexual tissue. Professor Gibson, with this 
in his imind, suggested that it might be possible by 
some means to induce the nuclei deficient in nuclein 
to absorb more, and so get back to the normal somatic 
condition. Farmer and others have shown that it is 
possible to induce such changes in the tissue of ferns, 
and for many months one of us (C. J. M.), acting on this 
knowledge, treated some cancer patients with nuclein, 
which was made by Professor Reynolds Green, but 
without proof that it conferred benefit. We, however, 
determined to experiment with it on individual cells. 1 

1 Quoted from a paper, " A Report on Cancer Research," by Dr. Macalister 
and myself, in The British M edicalJ ournal , October 23, 1909. 



EXPERIMENTS WITH NUCLEIN 179 

From the foregoing facts, believing that it was 
reasonable to suppose that chemical agents might 
influence human cell-division, we resolved to try the 
new in-vitro method. Bearing in mind that the cell- 
proliferation of healing appeared to be associated with 
the proliferation of cancer, our first step was to try the 
effect of nuclein on leucocytes. A saturated solution 
of it was made in " citrate solution, " and this was mixed 
with an equal volume of fresh blood. It was found 
that the nuclein seemed to lower the coefficient of 
diffusion of the cells very markedly compared with a 
control experiment in which no nuclein was employed. 
Some nuclein was then mixed up with jelly which 
contained stain and which had the right index of 
diffusion to stain leucocytes deeply, without killing them, 
in twenty minutes. But nuclein did not excite amoe- 
boid movements in the cells. 

In the next place some juice was squeezed from a 
malignant growth and citrated, and the citrated mixture 
was in its turn mixed with some fresh normal blood. 
It was found that this juice, like the nuclein, lowered 
the coefficient of diffusion of the leucocytes, but in 
addition it excited amoeboid movements in them. 

The lowering of the coefficient of diffusion due to 
nuclein was striking, because not only does the juice of 
a growth do the same thing, but the cells of cancer 
patients usually have a lowered coefficient. 

We were not satisfied, however, with this experi- 
ment with nuclein, because the preparation of it which 
we had obtained was very insoluble in neutral solution, 
and it was impossible to employ it in any more concen- 



180 DIVISIONS INDUCED IN LYMPHOCYTES 

trated form because more powerful solvents damaged 
or killed the cells. In place of this nuclein, therefore, 
extracts of some dead tissues were made, which we 
believed would contain the dead chromatin of cells, and 
it is said that chromatin contains nuclein. Moreover, 
it was thought advisable to keep as closely as possible 
to chemical substances which might be produced in the 
body, and the insoluble nuclein which we had used had 
been extracted by an elaborate process with hydro- 
chloric acid. 

To obtain this extract containing — as we believed — 
the chromatin of cells, we adopted a principle based on 
our observations of the phenomenon of achromasia. It 
may be recalled that achromasia is believed to be due 
to the chromatin of cells passing out of them, by 
dialysis, after their death. Achromasia will readily 
occur if cells are allowed to die in a solution which 
contains salt; and its onset after death is accelerated 
by heat. We therefore made an extract of a tissue by 
chopping it up in " citrate solution" and keeping it for 
twenty-four hours at 60° C. The first tissue chosen 
was lymphatic gland — the reasons for this being the 
knowledge that cancer frequently spreads through the 
lymphatic channels and glands, that lymphocytes are 
always seen in large numbers in growths, that lymphatic 
glands contain large numbers of lymphocytes, and 
especially because lymphocytes proliferate to a large 
extent when a tissue is chronically damaged. 

The small prevertebral (haemal) glands of lambs 
provided the lymphocytes whose chromatin we hoped 
to extract. These glands are composed almost entirely 



"PLIMMER S BODIES 181 

of lymphocytes. In the first instance a dilute extract 
was made in citrate solution, kept at 60° C. for twenty- 
four hours, and then filtered. Some fresh blood was 
mixed in a capillary tube with an equal volume of the 
filtrate and incubated at 37° C. for three hours. A drop 
of the mixture was then examined on the stained jelly 
which excites amoeboid movement in leucocytes (kinetic 
jelly). It was at once seen that the coefficient of 
diffusion of the leucocytes and lymphocytes had fallen 
remarkably — a greater fall than had ever been seen 
except that produced by morphine. The nuclei actually 
stained on this jelly in about fifteen minutes; and this 
jelly will never stain the nuclei of normal leucocytes — 
for they burst before that happens. It was also noticed, 
however, that the cells contained oval vesicles within 
their cytoplasm which closely resembled "Plimmer's 
bodies." After a while these bodies became identical 
with diffusion- vacuoles of large size, and when they 
burst some of them resembled archoplasm. It may 
be noted that other authors have suggested that 
"Plimmer's bodies" and archoplasm are identical. We 
think that these vesicles induced in leucocytes by the 
extract are diffusion- vacuoles due to the lowered 
coefficient of diffusion. 

The next series of experiments was made to observe 
the effects of this extract of haemal gland on leuco- 
cytes when the cells are spread on jelly which contains 
the extract. The jelly-films also contained the correct 
amount of Unna's stain to stain the granules of the 
cells, so that, if the extract had any action on the 
individual cells under these conditions, they would be 



182 DIVISION INDUCED IN LYMPHOCYTES " 

observed nicely stained and yet alive while this action 
was taking place. At first a dilute extract was used, 
as before, and the films in some instances were incu- 
bated for a short time, while others were suitably 
prepared for the room temperature. In one or two 
cases the lymphocytes seemed to contain some rod- 
shaped bodies in the cytoplasm. These rods stained 
a bright scarlet, similar to the staining of chromatin, 
and nothing had been seen like them before. They 
certainly were not bacteria, for we have often seen 
ingested bacteria which have quite a different ap- 
pearance; besides, they were only seen in the lympho- 
cytes, which we have never seen to ingest bacteria. 
With great hesitation we thought they might be 
chromosomes. 

Before proceeding farther it is necessary to explain 
that at the time when these experiments were made 
the appearance presented by the chromosomes of 
lymphocytes were unknown; in fact, it was not known 
whether these cells from the peripheral circulation 
divided by true mitosis or not. One of us had 
examined leucocytes by the in-vitro method for four 
years, and had never seen anything, previous to these 
last experiments, which appeared in any way connected 
with division of the white blood-cells. It was appre- 
ciated that, with the new method, a possibility existed 
that cell-division in white blood-cells might some day 
be seen; but to observe what appeared to be chromo- 
somes in lymphocytes, after we had tried only one or 
two groups of substances, seemed to be too good to be 
realised. It was necessary to be very careful, however,. 



LYMPHOCYTES MADE TO DIVIDE 183 

before we came to any conclusion as to the nature of 
the bodies which had been observed in the cells. 

The first striking point noticed about the red- 
staining rods was that they were not within the nuclei, 
but were in the cytoplasm outside the nucleus. This 
did not seem to be right, if the rods were chromosomes. 
It is usually considered that the phenomena of mitosis 
goes on within the nucleus as it does in plant-cells. 
Hitherto, mitosis in human cells, or animal cells gener- 
ally, had been seen only in cells which had been killed 
and fixed with heat or chemical agents at a time when 
they happened to be in the act of cell-division. From 
observation by the older method, it was usually under- 
stood that during mitosis the nuclear wall vanishes, 
and the chromatin within the nucleus forms into 
chromosomes, which then migrate into the cytoplasm. 
We were prepared to believe that the older methods 
might be fallacious owing to distortion caused by the 
killing and fixing of the cells, and to the fact that 
cells were only caught in the act of mitosis, not 
observed undergoing the whole phenomenon from start 
to finish. If our observations were correct, the rods 
in the lymphocytes were in the cytoplasm right enough, 
but the nuclear wall was still there internal to the 
chromosomes. 

The experimentation was then improved. Instead 
of the dilute extract being used, a concentrated one 
was made consisting of 50 grammes of haemal gland 
chopped up in 50 cc. {i.e. 100 per cent) of citrate 
solution, kept at 60° C. for twenty-four hours and then 
filtered as before. A jelly-film was made thus: To a 



184 DIVISIONS INDUCED IN LYMPHOCYTES 

tube of 5 cc. of "coefficient jelly," 0.5 cc. (5 units) of 
stain and . 8 cc. of alkali solution (8 units) were added, 
together with 3 cc. of the 100-per-cent extract, and the 
content of the tube was made up to a total of 10 cc. 
by 0.7 cc. of water. The jelly was boiled and a film 
made from it in the usual way, fresh citrated blood 
being placed on it. The object was to see whether this 
jelly would cause the rod-shaped bodies again to appear 
in the lymphocytes, for we believed that it was the 
extract which caused their appearance. It was neces- 
sary, therefore, to raise the index of diffusion of the jelly 
as high as possible short of killing the cells, in order 
to cause maximum diffusion of the contents of the jelly 
into the lymphocytes. The coefficient of diffusion of 
these cells is 14, and we added one more unit of alkali 
to the jelly in order to cause the extract to diffuse to 
the utmost into the cells. This is the equation : 

cf = (5s + 8a + 1.5x + Ih + t) - {Qc + 1.5n) = 15. 

where x is the 3 cc. of extract which is alkaline to the 
extent of about 1.5 units, and contains 3 per cent 
(3 units) of sodium citrate and 1 per cent (0.5 unit) 
of sodium chloride. 

Several fields of the specimen were first looked at 
and the ordinary resting condition of the lymphocytes 
noted. The slide was then placed in the 37° C. incubator 
for eight minutes. The same fields (containing the 
same lymphocytes) were then again examined, and 
pictures were seen which had never been seen before, 
for nearly every lymphocyte in the specimen was 



PHENOMENA OF MITOSIS 185 

unquestionably in the act of reproducing itself by 
mitosis. 

If any doubt existed as to whether the rod-shaped 
bodies which had been seen in the cytoplasm of the 
cells were really chromosomes, that doubt was now set 
at rest. The cells were certainly not reproducing them- 
selves when they were first placed on the jelly-film; 
but after they had absorbed the contents of the jelly 
during the eight-minutes' incubation at 37° C, they 
gradually went through the process of cell-division by 
mitosis, and on the removal of the slide from the 
incubator they were found in the act of reproduction 
with their chromosomes and centrosomes stained bright 
scarlet. 1 

These mitotic divisions, induced for the first time 
in living human cells, revealed the fact that the phe- 
nomenon of mitosis in lymphocytes differed in many 
details from the commonly accepted ideas regarding 
karyokinesis which have been adopted from the study 
(with the older fixation methods) of dead cells other 
than lymphocytes. The nucleus does not vanish; it 
forms the spindle. The chromosomes are not derived 
from within the nucleus, but are formed from the 
normal Altmann's granules which exist in the cyto- 
plasm. The centrosomes are not mere "dots" at the 
poles of the spindle, but are derived from the nucleolus 
which has divided into two. 

Fresh films were made, and bloods taken from other 



1 That division had been seen in lymphocytes with this jelly, and some of 
the facts which led up to this discovery were published by us in The British 
MedicalJournal, October 23, 1909. 



186 



DIVISIONS INDUCED IN LYMPHOCYTES 



persons were tried, and before long hundreds of mitotic 
figures were induced in lymphocytes, some of which 
closely resembled the karyokinesis, as described in 
the diagrams and drawings in well-known books on 
Cytology. The Altmann's granules, however, always 
form the chromosomes, 1 the nuclear wall forms the 
spindle, and the nucleolus forms the centrosomes. 
Thus: 



/ 






4 






7 



8 





10 




1 The chromosomes of lymphocytes do not always appear as definite 
"rods/' but may look as if they were composed of masses of granules. See 
photos. 



THE MITOSIS OF LYMPHOCYTES 187 

As will be shown in the succeeding chapters, one 
can now induce mitosis in lymphocytes whenever one 
pleases, and we have seen all stages of their cell-division. 
It must be remembered that to induce all these stages 
occupied many months of work, and involved the em- 
ployment of many varieties of the jelly-films. I shall 
now describe these divisions in detail, because we have 
since been able to induce divisions in other human 
cells, and therefore there is reason to believe that the 
phenomenon of mitosis in other varieties of cells is 
similar, if not identical, with that of lymphocytes, 
especially as regards the Altmann's granules forming 
the chromosomes and the nuclear wall forming: the 
spindle, both of which are important cytological 
points. 

The normal lymphocyte (figs. 40-2) occurs in a 
great variety of sizes in the body. In the blood 
one usually sees the smaller sizes, but in the glands 
(and not only in the lymphatic glands) the cell may 
reach large proportions. As will be shown later, it 
is quite a different class of cell, cytologiealry, from 
the so-called polymorphonuclear leucocyte, and it 
must spend only a portion of its life in the peripheral 
circulation. The lymphocyte has a large round or 
kidney-shaped nucleus, within which there are one or 
two nucleoli. In the living cell the nucleus appears 
to be a transparent membrane (fig. 40) which stains 
a faint blue with Unna's polychrome dye, 1 and it is 
tucked in at its poles to be attached to the nucleolus. 
Outside the nucleus, and studded on its surface, a 

1 Chromatin stains scarlet. 



188 DIVISIONS INDUCED IN LYMPHOCYTES 

large number of chromatin granules (figs. 41-2) are 
found which really are in the clear cytoplasm, and 
they are frequently extruded with the cytoplasm into 
the pseudopodia, especially if amoeboid movements are 
excited by atropine. When a lymphocyte "flagellates," 
these granules are thrust out through the cell-wall 
and become separated. When the cells are on jelly 
which makes them divide, amoeboid movements cease, 
and then the procedure is as follows: The nucleolus, 
which is shaped like a minute ring, and stains as if 
it was composed of chromatin, splits either into two 
rings 1 (figs. 43-4), or into two dots of chromatin 
which form the centrosomes. They then separate 
and emerge at opposite poles of the cell out through 
the mass of granules in the cytoplasm (figs. 45-7), 
and in doing this they seem to pull out the nucleus 
into the form of a spindle. The chromatin granules 
of the cytoplasm in the meantime are gradually 
collected into masses round the waist of the spindle 
(fig. 44), and ultimately they form a belt of 
chromatin round it on its outside (figs. 48-9). In 
a specimen in which one can see down through the 
spindle it will be observed that this belt divides into 
a number of chromosomes (figs. 50-1), which become 
semilunar-shaped with their points inwards (figs. 52-5) . 
Each chromosome is in contact with its neighbours 
at its points (figs. 62-3). Each one of them then 
divides into two (fig. 64). One half of every chro- 
mosome travels towards one nucleolus-centrosome, 

iOne of us (J. W. C.) has recently seen a ring-shaped centrosome in the 
act of division. It appeared hour-glass shaped. 



CHROMOSOMES OUTSIDE NUCLEUS 



189 





1 



A 



Fig. 4U. — A resting lympnocyte. J\ ote the deeply stained masses of gran- 
ules in the cytoplasm, which is bulged out in places. The large transparent 
nucleus and the stained ring-shaped nucleolus can also be seen. 







) 



i 






Fig. 41. — A resting lymphocyte. The Altmann's granules in the cyto- 
plasm are stained. 



CHROMOSOMES OUTSIDE NUCLEUS 191 






Fig. 42. — A resting lymphocyte. The cytoplasm, the granules, the nucleus, 
and the nucleolus can be distinguished. 





Fig. 43. — The earliest stage of mitosis. The nucleolus has divided into two 



CHROMOSOMES OUTSIDE NUCLEUS 



193 




Fig. 44. — Early mitosis in a lymphocyte. Looking down through the 
spindle (polar aspect). The nucleolus has divided into two centrosomes, 
each of which is ring-shaped. The spindle is surrounded by a belt of chro- 
matin granules. 




Fig. 45. — Mitosis in a lymphocyte. Profile aspect. The two ring- 
shaped centrosomes can just be seen towards the poles. The granules are 
becoming formed into chromosomes. 

*3 



CHROMOSOMES OUTSIDE NUCLEUS 



195 




Fig. 46. — Foreshortened appearance of a mitotic figure in a lymphocyte. 
The position of one nucleolus-centrosome at the pole of the figure is well 
shown. 




Fig. 4' 



-Profile aspect of mitosis in a lymphocyte. The relative positions 
of the centrosomes and chromosomes can be seen. 



CHROMOSOMES OUTSIDE NUCLEUS 



197 



Fig. 48. — Profile aspect of mitosis. The belt of chromatin is formed round 
the waist of the cell. 




Fig. 49. — One resting and one dividing lymphocyte. In the latter the 
chromosomes are beginning to divide. The centrosomes appear as dots 
of chromatin. 



CHROMOSOMES OUTSIDE NUCLEUS 199 



Fig. 50. — Polar aspect. The belt of chromatin granules is dividing into 



chromosomes. 








Fig. 51. — Polar aspect. The chromosomes are becoming semicircular. 



CHROMOSOMES OUTSIDE NUCLEUS 



201 



^ 



Fig. 52. — Polar aspect. An "aster" stage of mitosis in a lymphocyte. 




Fig. 53. — Polar aspect. Some of tne chromosomes are semicircular-shaped; 
some are dots of chromatin. 



CHROMOSOMES OUTSIDE NUCLEUS 203 






Fig. 54. — Polar aspect. One centrosome can be seen at the pole of the 

"aster" figure. 




Fig. 55. — Polar aspect. Sixteen chromosomes could be counted in this cell. 



CHROMOSOMES OUTSIDE NUCLEUS 205 





Fig. 56. — Profile aspect of mitosis in a lymphocyte. 











Fig. 57. — Profile aspect of mitosis in a lymphocyte. 



CHROMOSOMES OUTSIDE NUCLEUS 207 




,/ 





Fig. 58. — Profile aspect. The chromosomes can be seen at the waist of the 

spindle. 




Fig. 59. — Profile aspect. A figure frequently seen. 



CHROMOSOMES OUTSIDE NUCLEUS 209 




Fig. 60. — Profile aspect of mitosis. 



r 



. 



x. ■ . : ■ ,: 





Fig. 61. — Oblique aspect of mitosis in a lymphocyte. 




U 



CHROMOSOMES OUTSIDE NUCLEUS 



211 




Fig. 62. — Polar aspect of mitosis in a large lymphocyte from a patient 
suffering from carcinoma. There are sixteen chromosomes. 




Fig. 63. — Polar aspect. The chromosomes were V-shaped with their 
apices inwards to be attached to the nucleus-spindle, which can dimly be 
made out. 



CHROMOSOMES OUTSIDE NUCLEUS 213 




Fig. 64. — Polar aspect of mitosis in a large -lymphocyte from a cancer 
patient. The chromosomes are dividing. 





Fig. 65. — Profile aspect of mitosis. 



CHROMOSOMES OUTSIDE NUCLEUS 215 




Fig. 66. — Profile aspect. The figure is fuHy formed. One nucleolus- 
centrosome is ring-shaped; the other is a dot of chromatin. 








Fig. 67. — Profile aspect. The sixteen chromosomes could be counted. 



CHROMOSOMES OUTSIDE NUCLEUS 217 





Fig. 68. — The cell has become constricted in its centre. 





Fig. 69. — Profile aspect. Complete division is about to occur. The 
chromosomes are being reconverted into granules, but the mitotic figure is 
not quite finished at the dividing-point. 



CHROMOSOMES OUTSIDE NUCLEUS 



-219 




Fig. 70. — Profile aspect. The spindle and chromosome 
the cell wall-has not yet separated. 



have divided, but 




Fig. 71. — Completion of mitosis in a lymphocyte. 



CHROMOSOMES OUTSIDE NUCLEUS £21 

and the other half towards the other centrosome 
(figs. 56-61, 65-7). The spindle divides in the centre 
(figs. 68-70); and lastly the cell itself divides (Hg. 71). 
In each daughter cell the chromosomes return to 
their granular condition and pervade the whole 
cytoplasm. The single centrosome (for there is now 
one only in each daughter cell) again becomes tucked 
into the centre of the transparent nucleus — which 
consists of one half of the original spindle, and thus 
the cycle of mitosis is completed. Doubtless each 
chromosome granule divides during some part of the 
cycle, but owing to their minute size we have not 
been able to see their division. 

The cells, of course, do not usually divide in 
definite stages such as the aster and diaster, although 
sometimes a cell will be found which presents one 
of them. Sometimes one sees that the chromosomes 
may be dividing in one part of the cell, while some 
chromosomes in another part are being reconverted 
into granules of chromatin. The way in which a 
cell is lying on the jelly must be taken into con- 
sideration in the determination of the stage of mitosis. 
One rarely finds a perfect figure as described in 
diagrammatic drawings of other types of cell, for the 
cells frequently appear foreshortened owing to the 
oblique manner in which they happen to come to 
rest under the cover-glass. The position of the ring- 
shaped nucleolus-centrosomes is of prime importance 
in the determination of the stage of the mitotic 
figure. 

In observing any stage of mitosis, however, it will 



222 DIVISIONS INDUCED IN LYMPHOCYTES 

be seen at once — a point on which we must lay stress 
— that the chromosomes are outside the nucleus 
are formed by the conglomeration of the Altmann's 
granules in the cytoplasm. As will be shown later 
on, this is also the rule in leucocytes and some epithelial 
cells as well as in lymphocytes. 

The phenomenon of mitosis, then, as seen in these 
cells when they are stained alive, differs very materially 
from the usual descriptions of it as seen in cells which 
have been killed, fixed, cut into sections or otherwise 
manipulated, and stained. The old idea was — although 
divisions had not been seen actually in lymphocytes — 
that the chromosomes were formed out of some 
chromatin which is within the nucleus, and that, inside 
this again, a spindle, which does not exist in the rest- 
ing stage, is formed. The nuclear wall was described 
as vanishing during mitosis according to most con- 
ceptions. But, as I have described, mitosis is a much 
simpler phenomenon. 

The misconception has been due, I think, to several 
factors. In the first place most cytological research has 
been carried out with plant-cells, and animal cytology 
has arisen from it. In the second place, cells up to now 
have only been caught in the act of mitosis; their cycle 
of cell-division has not been followed from the resting 
stage to completed division in one individual cell. The 
morphological elements of a resting cell have been 
studied, and then those in one killed in the act of 
division, and the part played by each element has been 
deduced from its new position — not watched through- 
out. Lastly, owing to manipulation, the so-called 



altmann's granules 223 

Altmann's granules of some cells have been crushed 
into the nucleus, in which case they look as if they 
formed part of it, or were inside it — a fallacy which has 
given rise to great controversy regarding the nature of 
these granules, to the statement that they do not exist 
in some cells, e.g. lymphocytes and cancer cells, and to 
failure of appreciation of the fact that the chromosomes 
are formed out of them. Let mitosis be induced in 
a living cell and no second glance will be required 
to realise the real sequence of events. 

The mitosis of plant-cells seems to go on within 
the nuclear wall, but this is not the case in the animal 
cells which we have seen. The granules in the cyto- 
plasm of Altmann's granules are larger in some classes 
of cells than in others. For instance, they are much 
larger in eosinophile leucocytes than in lymphocytes. 
When they are large their position is obvious, but when 
they are small, as in lymphocytes and cancer-cells, 
during the killing of the cell as it is fixed the small 
granules — which are composed of chromatin — adhere 
to and are merged into the nucleus. No matter how 
we try to fix a specimen, death takes time, and the 
liquefying cytoplasm bulges out the cell- wall. The 
rapidity of death depends upon the diffusion of the 
fixative into the cell, and this diffusion takes time. 
Hence when a cell is stained and fixed, it appears as 
if its nucleus is a mass of chromatin — which it 
is not — and its halo of cytoplasm, which has bulged 
out of the cell- wall, is now apparently devoid of 
Altmann's granules. This is a pitfall into which we fell 
ourselves, for, although we had seen the granules of 



224 DIVISIONS INDUCED IN LYMPHOCYTES 

lymphocytes and cancer-cells forming the chromo- 
somes, we thought that these granules were composed 
of chromatin, but that Altmann's granules as ex- 
emplified in polynuclear leucocytes were of quite a 
different nature. As will be shown later, the granules 
of leucocytes also form the chromosomes in the same 
way as those of lymphocytes and cancer-cells. Professor 
Lorraine Smith suggested that the apparent absence of 
granules in lymphocytes and cancer-cells might be due 
to the fixative, and he is right. The cytoplasm of every 
living lymphocyte is full of minute granules which stain 
like chromatin with aniline dyes, and these granules 
clump together to form the chromosomes during cell- 
division — a point about which there can be no question 
whatever. 

In some other cells, such as some large cells of the 
liver, we have seen large granules in the cytoplasm (as 
well as fat globules), which will not stain. What their 
function is we do not know, for we have not been able 
to induce divisions in these cells. The granules of 
lymphocytes we shall henceforth style "chromosome- 
granules," the nucleolus as the "nucleolus-centrosome," 
and the nucleus as the "nucleus-spindle." 



CHAPTER XI 

THE DIVISION OF LYMPHOCYTES INDUCED BY THE ANILINE 

DYE THE AUGMENTING ACTION OF ATROPINE AND 

EXTRACT OF HAEMAL GLAND "aUXETICS" THE 

CYCLE OF CELL-DIVISION THE POSSIBILITIES OF 

THE INDUCED CELL-DIVISION BEING DUE TO 
"DEATH-STRUGGLES" ASYMMETRICAL AND RE- 
DUCED DIVISIONS 

The fact that mitotic figures could be made to appear 
in lymphocytes was very satisfactory, for it seemed to 
us to be a step in the solution of the problem of the 
cause of the multiplication of cells. It was true that 
we had only seen them in lymphocytes; but still these 
mitotic divisions had occurred in response to the action 
of a chemical substance, and if these cells were capable 
of dividing in response to it, it appeared reasonable to 
suppose that other cells would do the same and that 
it was possible that they would only divide when they 
absorbed a chemical substance. We believed at first, 
of course, that the substance which had induced the 
divisions was the extract of the dead haemal gland; 
but before many experiments had been made this 
suggestion received a check. One day a cell was seen 



226 THE CYCLE CELL-DIVISION 

stained in an early stage of mitosis; its ring-shaped 
nucleolus-centrosome was lying at a pole of the nucleus- 
spindle in the cytoplasm, outside the mass of granules 
which had not yet collected round the waist of the cell. 
Now when this early figure was seen by me, I remem- 
bered that I had seen something very similar to it before, 
and on turning up a paper (British Medical Journal, 
January 16, 1909), which described some work done 
more than a year previously, it was found there men- 
tioned that the nucleoli sometimes appeared outside 
the nucleus in the cytoplasm. Now, this position of 
the nucleolus-centrosome is the first step in mitosis, 
and therefore it was grasped that this mitosis must have 
been seen before, although the fact was not realised at 
the time. Another far more important point was also 
grasped, viz. that when the mitosis had been seen a year 
previously, no extract of haemal or any gland had been 
either used or thought of. 

The notes of the previous work were referred to, 
and it was found that when the — as it turned out — 
early stage of mitosis had been seen, the cells had been 
resting on a jelly which contained only Unna's stain 
and atropine. It was clear, therefore, that either one 
or both these substances would induce divisions in 
lymphocytes and our hopes were rather damped, for 
both these substances, unlike the extract of haemal 
gland, are entirely artificial, and could not possibly be 
concerned in the cell-proliferation of healing. 

Each of the ingredients of the jelly — described in 
the last chapter — which induced well-marked mitosis 
in lymphocytes was now tried separately. Jellies were 



MITOSIS DUE TO THE DYE 227 

prepared which contained each of them in turn, and 
jellies were prepared which contained only the salts 
sodium citrate and sodium chloride. Many experi- 
ments were made from each, and several different 
strengths of the different substances were tried re- 
peatedly on fresh lymphocytes. It was thus ascertained 
that Unna's polychrome methylene blue (Grubler) con- 
tains some substance which will induce divisions in 
lymphocytes. It requires a high concentration of this 
stain for this purpose, and this was the reason why 
advanced divisions had not been seen in the several 
years' previous work with this dye. Unless the jelly 
contained Unna's stain, no mitosis whatever would 
occur. Repeatedly they were tried, but none of the 
other ingredients by themselves could be made at that 
time to cause lymphocytes to reproduce themselves. 
The 100-per-cent extract of haemal gland by itself 
certainly did not do so, nor did the atropine; but 
both the extract and atropine — and this was an im- 
portant point — greatly augmented the action of the 
stain in inducing mitosis. By itself at least 10 units 
(1 cc.) of polychrome stain were required to induce 
mitosis; but if a certain quantity of atropine or of 
extract, or, better still, of both, was also added to the 
jelly, one could cause advanced mitosis in lymphocytes 
with only two or three units of stain. It was a 
remarkable state of affairs that neither atropine nor 
extract would induce divisions by themselves, but that 
they augmented the action of the stain in doing so to 
a very marked degree. 

During all this experimentation, which occupied a 



228 THE CYCLE OF CELL-DIVISION 

considerable time, many points connected with the proc- 
ess of inducing divisions were learnt. We had three 
factors to deal with, viz. polychrome dye, atropine, and 
extract consisting of the soluble remains of dead haemal 
gland of 100 per cent. It was found that lympho- 
cytes would not make any attempt whatever to divide 
unless they absorbed some of the polychrome stain. As 
the stain passed into the cells, it stained first their 
chromosome-granules and their nucleolus-centrosomes. 
Like polynuclear leucocytes, lymphocytes do not appear 
to suffer much harm to their lives while their granules 
are stained, but as soon as their nucleolus-centrosomes 
are reached by the dye death occurs. Mitosis takes 
place about the time when the granules are staining, 
and therefore the rapidity of the onset of mitosis depends 
on the rapidity of the diffusion of the dye into the cells. 
It is thus evident that the gradual diffusion of the 
stain first causes mitosis and then death because it 
kills the cells by combining with and staining the 
nucleolus-centrosome. The rapidity of the diffusion 
of the stain is increased by concentrating it, by the 
presence of alkalies, or by heat. These factors also 
hasten death and they likewise hasten cell-division. 
With regard to the factor heat, however, we must add 
the qualification that no lymphocyte will divide below 
a temperature of 30° C. or above about 40° C, and for 
this reason we have employed a temperature of 37° C. 
throughout these experiments for inducing division. 

Now, mitosis is a process which occupies a certain 
amount of time. If the diffusion of the stain is 
very slow, the time taken by the act of mitosis is 



TIME TAKEN BY ACT OF MITOSIS 229 

correspondingly slow. But as far as we can see, mitosis 
cannot occur completely in less than about three 
minutes. It can take a very long time in its accom- 
plishment ; but it cannot be completed in less than three 
minutes. Hence, if mitosis can take place slowly, 
without the cell being killed by the stain, complete 
mitosis can occur; but if the nucleolus-centrosome 
stains in less than a minute or so, death will occur 
before the cell has had time to divide. This fact 
governs the whole of this experimentation, for when 
inducing cell-division with the aniline dye it must 
be remembered that the mitosis has to occur after the 
cell-granules have begun to stain, but before death is 
occasioned by the staining of the nucleolus-centrosome. 
We have the power of accelerating and delaying the 
diffusion of the stain into the cells by adding or sub- 
tracting alkali, or by increasing or decreasing the 
concentration of the stain by rules which can be plotted 
in an equation, and therefore by such an equation we can 
ascertain the rate of cell-division as induced by the 
chemical agent. But throughout it must be appre- 
ciated that it is the stain which is inducing the 
cell-division, and that if the stain is not sufficiently 
concentrated no division will occur at all. On the 
other hand, it must also be remembered that an excess 
of stain will poison the cells too quickly. A cell must 
absorb a certain amount of stain before it will divide, 
and the absorption depends on the concentration of the 
stain in the jelly and on the alkali. One may place 
living blood-cells on a jelly which contains the best 
ingredients for inducing cell-division; but unless the 



230 THE CYCLE OF CELL-DIVISION 

alkali is correct according to the equation — that is to 
say, unless the index of diffusion of the jelly is correct 
for the coefficient of diffusion of the cells, the latter 
will take no notice whatever of the mitosis inducing 
agents in their surroundings. But make diffusion 
factors of the jelly right and the cells will then re- 
spond immediately, and as many as 90 per cent of 
the lymphocytes in a specimen may be made to divide. 
Not only does the rapidity of the onset of mitosis 
depend on the physical laws of the diffusion of sub- 
stances into cells, but the actual stage reached in a 
given cycle of cell-division also depends on them; for 
the completion of the mitotic cycle occupies a certain 
amount of time, which varies inversely with the quantity 
of the stain absorbed by the cell, and this absorption 
depends on the coefficient of diffusion, heat, alkali, etc. 
The following experiment illustrates this point. A 
jelly-film was made which induced almost completed 
divisions in lymphocytes in ten minutes. By making 
several films and removing them, one at a time, 
from the 37° C. incubator at each minute, it was 
seen that mitosis began with the staining of the 
granules at about the seventh minute, and that death 
occurred at about the ninth. The experiment was 
repeated, and at the seventh minute, immediately while 
mitosis was occurring, the slide was quickly removed 
from the 37° C. incubator to one which maintained 32° C. 
The sudden lowering of the temperature delayed the 
diffusion of the stain into the cells, and the interesting 
point is that the mitosis ceased when the diffusion of 
the stain was suddenly arrested, and the cells died 



INFLUENCE OF VITALITY 231 

slowly. Twenty minutes afterwards, when all the 
chromatin was stained, it was seen that the mitosis 
had been arrested in those early stages reached at the 
seventh minute. 

Thus it appears from this experimentation that not 
only will a lymphocyte not reproduce itself in vitro 
unless it absorbs a chemical "exciter of reproduction," 
but also the actual stage reached in its act of mitosis 
varies directly with the quantity of that substance 
which has diffused into the cell. It follows that, 
in vitro, before a cell will reproduce itself completely 
it must receive a definite quantity or dose of the 
chemical substance. 

In addition to the above factors, the divisions of the 
cells depend upon their vitality. If some blood is 
citrated and kept for two days, it is very difficult to 
induce divisions in the lymphocytes. The longer cells 
have been shed the slower they are to respond to the 
division-inducing action of the stain, in spite of the fact 
that their coefficient of diffusion has fallen. It is im- 
possible to induce divisions in cells with auxetic jelly if 
other cells from the same sample of blood will not show 
excited movements on kinetic jelly. 

The foregoing points showed that the reproduction 
of lymphocytes in vitro depended entirely on the 
aniline dye. The dye did not merely increase the cells' 
propensity to divide; it actually caused the division. 
Lymphocytes had never been seen to divide before, and 
they certainly will not divide in vitro unless one takes 
deliberate steps to make them do so. Mitosis is a 
complex phenomenon which only occurs as an act of 



232 THE CYLE OF CELL-DIVISION 

cell-reproduction, and in vitro the only way to cause 
it to take place was to force the cells to absorb the 
chemical " exciter of reproduction" contained in the 
aniline dye. It appeared reasonable to us to suppose 
that there might be other "exciters of reproduction," 
not only for lymphocytes, but for other cells as well, 
and therefore we proposed to call the substance in the 
aniline dye which caused cell-division in lymphocytes 
an "auxetic" (av^rucos, an exciter of reproduction), 
a convenient term suggested by Professor Harvey 
Gibson, which might be applied to other substances 
having a similar action if such were proved to exist. 

The next steps were to investigate the "augment- 
ing" actions of both atropine and the extract of haemal 
gland. It has been pointed out how atropine, being 
an alkaloid, greatly excites amoeboid movements in 
lymphocytes and leucocytes, and it was soon seen that 
atropine also greatly augments the action of the poly- 
chrome dye in inducing mitotic figures in lymphocytes. 
The best strength of atropine to be added to the jelly 
which contains the stain is that which causes maximum 
excitation of amoeboid movements. If this is done 
lymphocytes can be caused to divide with the strength 
of the stain reduced to one-fifth of the minimum 
amount of it which will, by itself, induce mitotic 
figures. In other words, atropine will not by itself 
induce divisions on the microscope slide, but it will 
augment the "reproducing" action of polychrome stain 
five-fold. Another point was also noticed, which was 
very material to the main object of these researches, 
in that stain, plus atropine, caused lymphocytes to 



STAIN ACTS ON THE GRANULES 233 




Fig. 72. — Asymmetrical mitosis in a lymphocyte induced by azur stain 
augmented by atropine. 




Fig. 73. — Asymmetrical mitosis induced by azur stain augmented by 

atropine. 



STAIN ACTS ON THE GRANULES 235 

undergo curious one-sided mitoses in some instances 
(figs. 72, 73). 

We now investigated the " augmenting" action of 
the extract of haemal gland. This was even more 
powerful than that of atropine. So great was it that 
one can employ a jelly which only contains three units 
of polychrome stain — which will never induce divisions 
by itself; and if 3 cc. of the 100-per-cent extract of 
dead haemal gland is also contained in the jelly, complete 
divisions can be induced in lymphocytes without the 
cells actually being coloured by the stain at all. Yet 
all attempts at this stage to cause the extract to induce 
divisions by itself had failed. 

Thus, by means of a mixture of a little stain, say 
4 units, . 7 cc. of a 1-per-cent solution of atropine 
sulphate, 3 cc. of the 100-per-cent extract of haemal 
gland, 6 units of alkali solution, and 0.3 cc. of water 
added to 5 cc. of coefficient jelly to make a total of 
10 cc, one can induce advanced divisions in lympho- 
cytes, without the cells staining at all in ten minutes. 

cf = (4s + 6a + 1 . 5x + Ih + 1) - (6c + 1 . 5n) + . 5a) = 11 . 5. 

We have already stated that mitosis occurs about 
the time when the stain has diffused into the cells 
sufficiently to stain the granules. But now with the 
combination of stain and the augmenting substances 
mitosis will occur without the stain colouring the 
granules at all. In spite of this, however, stain is 
essential. Hence we suggest the theory that the 
stain induces divisions by acting on the chromosome 



^36 THE CYCLE OF CELL-DIVISION 

granules; but that, since it is not necessary for it 
actually to colour these granules, as shown by the last 
experiment, it seems probable that the stain induces 
divisions by virtue of some substance contained in it 
which does not colour granules. It is not the stain 
itself which induces divisions; it is some constituent 
of it, and the action of that constituent is greatly 
augmented by atropine and extract. 

The next point is that when mitosis is induced on a 
microscope slide with stain, death is premature. Even 
if there is not sufficient stain to colour the nucleolus- 
centrosomes, death rapidly follows. We believe that 
this dye contains at least two constituents which can 
be utilised differently by the cell's protoplasm — a 
substance which, by combining with the cell-gran- 
nies, causes the cell to reproduce itself, and a poison 
which kills it. Both diffuse into the cell together; 
mitosis is induced and then the cell dies prematurely. 
If the stain is sufficiently concentrated, the chromatin 
after it is dead will combine with it, and the chromatin 
then turns bright scarlet. From prolonged observation 
of these induced divisions we think that the scarlet 
coloration of the chromatin is a post-mortem effect. 
The stain as it diffuses into the cell induces division as 
it combines with the granules, which die and become 
coloured one by one. All the time the stain is passing 
farther into the cell, and later and later stages of mitosis 
are being induced. Ultimately the nucleolus-centro- 
some is reached and the cell dies; and thus it is seen 
dead in the act of mitosis with its chromosomes and 
centrosomes stained bright scarlet. If, on the other 



THE "azur principle 237 

hand, the concentration of the stain is reduced and its 
action augmented by atropine and extract, still the 
poison, but in less strength, passes into the cell; and 
although mitosis occurs to an advanced degree, never- 
theless premature death occurs in spite of the fact that 
there is not sufficient strength of colouring matter to 
give rise to the post-mortem coloration of the chromo- 
somes and centrosomes. Death is a gradual process — 
presumably it is molecular as well as cellular, for the 
post-mortem scarlet coloration occurs gradually; but 
it is not until the nucleolus-centrosome is reached 
that all mitosis ceases. One cannot excite amoeboid 
movements in a cell which has its nucleolus stained. 

Since Unna's polychrome methylene blue contained 
the active principle which caused the cells to divide, 
and the other two substances appeared merely to be 
augment ers, we now turned our attention more es- 
pecially to the dye. Polychrome methylene blue stains 
chromatin scarlet and the nucleus-spindle a faint blue. 
It is made by " polychroming " methylene blue. Fresh 
methylene blue stains chromatin blue, and it is not so 
effective as the polychrome dye in inducing mitosis. 
The "polychroming" process consists of rendering 
a solution of methylene blue alkaline with sodium 
carbonate and naturing it for some time at a high 
temperature. The methylene blue turns a purple 
colour. This is due to decomposition — an oxidation 
occurs with the production of a dye known as "azur." L 
This azur dye can be obtained from dealers, and it 
can be extracted from the polychrome dye by means of 

1 Centralblattfiir Bakteriologie, bd. xxix., 1901, p. 765. 



238 THE CYCLE OF CELL-DIVISION 

chloroform. It was found that the constituent which 
induces divisions in lymphocytes is almost entirely 
confined to the azur dye. The more the azur was 
extracted the less efficient the polychrome dye became, 
and the azur is very potent although it does not stain 
the chromatin as well as the polychrome dye. 

A concentrated solution of azur dye was made 
thus : In a burette 20 cc. of Unna's polychrome methy- 
lene blue (Grubler) had added to it 20 cc. of chloroform, 
and the mixture was allowed to stand for 12 hours. 
The chloroform, which sinks to the bottom, carrying 
some of the azur dye with it, was then run off into 
a shallow dish, where it was allowed to evaporate. 
20 cc. more of chloroform was then added to the 
original 20 cc. of stain in the burette, and, after 
12 hours, it, in its turn, was run off into the same 
dish and also allowed to evaporate. This procedure 
was repeated five times, and the dry azur dye was 
so obtained. Lastly, a solution of this dye was made 
by adding 5 cc. of water to the dish. This potent 
dye is a fluorescent red one, which, when dry, shines 
with a metallic lustre. A very powerful jelly for 
causing mitotic divisions in lymphocytes was made by 
substituting . 4 cc. of this potent solution for the . 2 cc. 
of Unna's stain in the last equation. By means of this 
jelly all stages of divisions can be readily obtained, it 
being only necessary to vary slightly the content of 
alkali in producing early or late mitosis in the ten 
minutes. It is better to keep at least two units of 
polychrome stain in the jelly, in order to stain the 
chromosomes more deeply. 



REDUCTION DIVISIONS 239 

In experimenting with this last jelly containing 
azur dye, an important point was found out. By 
delaying death as long as possible, by employing the 
minimum amount of alkali which will make the cells 
undergo mitosis, we at last succeeded in keeping the 
lymphocytes alive for twenty minutes, and yet mitosis 
was being induced during the greater part of the time. 
That is to say, mitosis was induced as early as pos- 
sible, for, as will be shown in the next chapter, we 
cannot, under the experimental conditions, keep up 
the cell's vitality longer than twenty minutes, and it 
is difficult to keep them alive to divide for a longer 
time than even ten minutes; still their lives have been 
prolonged for twenty minutes. 

The point revealed by this experiment was that 
the so-called reduction division is not a special form 
of mitosis in lymphocytes. The somatic number of 
chromosomes in the body is thirty-two, but hitherto 
in all the dividing lymphocytes in which it was pos- 
sible to count the actual number of chromosomes their 
number was either sixteen or thereabouts (figs. 62, 63, 
67). In other words, the divisions which we induced 
with the stain in ten minutes were of the reduced 
variety, or what Farmer, Moore, and Walker called 
"maiotic" divisions. By prolonging life, however, for 
twenty minutes, and inducing the divisions slowly, 
especially if only the early stages of mitosis were 
induced in the time, it was found that now lympho- 
cytes divided by somatic divisions with more than 
sixteen and sometimes with a full number of thirty- 
two chromosomes (figs. 74, 75), and the statement 



240 THE CYCLE OF CELL-DIVISION 

that the wandering cells of the body only divide by 
reduced divisions of the " reproductive" type is thereby 
disproved. In lymphocytes examined on a microscopic 
slide the question of the number of chromosomes seems 
to be entirely one of degree— it depends on the rapidity 
of the division, which, in its turn, depends on the 
quantity of the "auxetic" absorbed by the cell. By 
increasing the alkali one can induce divisions very 
quickly, provided of course there is not too much 
alkali. We have seen lymphocytes divide with less 
than sixteen chromosomes, and on one occasion, when 
mitosis was very rapidly induced, the number was 
reduced to eight only; but the number of chromo- 
somes seems usually to remain in these round numbers, 
namely, thirty-two, sixteen, eight, the last one being 
very rare. If a division is induced in the usual way 
with a jelly which will kill the cells in about ten 
minutes, the number of chromosomes is nearly always 
sixteen, but a slow division will be a somatic division. 
There does seem, however, to be a difference in the 
way the chromosomes split. We have seen them in 
the act of splitting longitudinally, and also, and more 
commonly, they split transversely; although whether 
the longitudinal splitting is significant of a "first 
(heterotype) maiotic" division or not we are not in 
a position to state. 

The asymmetrical mitoses induced when atropine 
is present, especially if it is present to excess, are 
interesting. The mitosis seems to be going on in 
one side of the cell. We have not seen a completed 
division in one of those asymmetrical mitoses, but we 



FACTS SUMMARISED 



241 






Fig. 74— An early stage of delayed mitosis induced by a jelly with a low 
index of diffusion. The number of chromosomes is more than sixteen. 




Fig. 75. — Thirty-two chromosomes could be counted in this cell. Early 

mitosis delayed. 

16 



FACTS SUMMARISED 243 

think that, from the appearance of the cells, they are 
about to divide into more than two daughter cells by 
some quite atypical arrangement of the chromosomes. 
The point is a very important one, for asymmetrical 
divisions are reported to be frequently seen in can- 
cerous growths. 

We may now summarise the facts learnt from the 
mitotic divisions induced in lymphocytes by the aniline 
dye. (1) Lymphocytes will not divide in vitro unless 
they absorb the chemical agent. (2) The rapidity of 
the onset of division depends on the rapidity of the 
diffusion of the agent. (3) The time occupied by the 
act of division depends on the amount of the agent 
absorbed and the time occupied in the diffusion of the 
substance into the cell. (4) If the diffusion is slow 
the cells divide with the somatic number of thirty- 
two chromosomes; but if it is rapid the number is 
reduced. (5) A "reduction division" means that a 
cell is very prolific, owing to its absorption of a large 
quantity of the chemical agent. (6) The rapidity of 
the absorption of the agent depends on the coefficient 
of diffusion of the cell, the concentration of the agent 
in the surrounding fluids, and on the presence and 
strength of the factors which increase or decrease the 
diffusion of the substances. (7) Lastly, it depends on 
the vitality of the cells themselves. In fact, the 
division of lymphocytes on a microscope slide depends 
entirely on the presence or absence of a chemical 
agent, and, if it is present, on its strength and on its 
diffusion into the cell. 

In our opinion, judging from the mitotic figures 



244 THE CYCLE OF CELL-DIVISION 

which have been induced in cells, mitosis should not 
be described as the phenomenon of nuclear division. 
It is part of the cycle of cell-division, and the whole 
of the cell-protoplasm takes part in it. The Altmann 's 
granules form the chromosomes, the nucleolus forms 
the centrosomes, and the nucleus forms the spindle. 
The protoplasm of the cytoplasm and cell-wall also 
reproduces itself and divides during mitosis. 

The active principle in the stain which causes 
mitosis in lymphocytes is a constituent of the azur 
dye. This dye also contains a substance which kills 
the protoplasm, and having done this it will, if in 
sufficient concentration, cause that protoplasm to stain 
scarlet. Mitosis occurs by the action of the active 
principle on the chromosome granules; cell death 
occurs by the action of the poison on the centrosomes. 
So far the active principle has proved to be inseparable 
from the poison in the anilin dye. 

Up to this stage in the researches the only sub- 
stance we had found which would induce divisions in 
lymphocytes was this anilin dye; but its action was 
augmented by atropine and an extract of dead haemal 
gland. Atropine augments its action five-fold if it is 
absorbed in suitable strength, in which case it may 
induce asymmetrical mitosis. Neither atropine, in no 
matter what strength, nor extract of dead haemal gland 
in the strength of 100 per cent will by themselves 
induce mitotic figures in lymphocytes. 

Great care must be exercised in the practice of 
inducing the mitotic figures in lymphocytes. The 
jelly must be accurately prepared, but it is better to 



TECHNIQUE 245 

allow it to be deficient in alkali by a unit or so at 
first. A film is made with some fresh blood spread 
on it and incubated for ten minutes. The temperature 
of 37° C. must be accurate; many failures have resulted 
owing to the neglect of regulation of the incubator 
temperature. If the chromosome-granules of the 
lymphocytes are unstained, a drop or two of alkali 
solution is added to the jelly and a fresh film is tried. 
Soon the right alkalinity will be obtained to induce 
early mitosis in the ten minutes ; and now if more alkali 
is very carefully added to the jelly and another film 
made, later stages of mitosis will be induced. It is 
instructive to proceed farther and once more add alkali, 
when the cells will be killed too quickly, and only very 
early stages will be seen in the ten minutes, for there 
has not been time for late phases to occur before the 
cells have died. If more alkali is again added, owing 
to rapid death the cells will appear quite at rest, as if 
there had been no agent to cause cell-division in the 
jelly at all. But the granules and nucleoli will be 
deeply stained, and the polymorphonuclear cells will 
probably all be burst and achromatic. 

When first we showed the mitotic figures to some of 
our friends we received some adverse criticisms. It is 
always possible to induce mitosis in lymphocytes, but it 
is not always possible, at a few minutes ' notice, to find 
figures which resemble the diagrammatic drawings of 
mitosis in the cells of plants and the lower animals as 
given in the text-books on cytology. However, when 
a convincing figure did appear, the nature of the 
chromosomes, the spindle, and the centrosomes were 



246 THE CYCLE OF CELL-DIVISION 

immediately appreciated. We, of course, maintained 
that the divisions were induced by a specific chemical 
substance contained in the stain, pointing out that 
lymphocytes had never been seen to divide before, and 
that mitosis will only occur in them if they absorb a 
certain quantity of the substance. But our friends 
"one and all began to make excuse." Some said that 
the divisions were in the nature of a death-struggle; 
they pointed out — a fact which we admitted — that 
death always was premature, and it usually occurred 
during the act of mitosis. We explained the cause of 
death, but still the suggestion of the "death-struggle" 
was maintained by some in the absence of proof 
against it. 

Others suggested that the divisions were entirely 
artificial and not at all like the natural method of cell- 
proliferation, although they had never seen the latter. 
We admitted the fact that at this stage of our re- 
searches we could only induce divisions in lymphocytes, 
and we could only do this with an entirely artificial 
anilin dye; but still we found it difficult to appreciate 
why a cell should go out of its way to divide by an 
entirely abnormal process. We suggested that we 
thought that if a cell was going to divide at all, it 
would try to do so by the normal process to which 
it was accustomed. But the suggestion that the mitotic 
divisions were "freaks" remained to be disproved. 

It appeared to us a remarkable thing that a cell 
should try to reproduce itself by cell-division in a 
death-struggle; it seemed such a futile thing for it 
to do. Moreover, other stains — such as ordinary 



TECHNIQUE 247 

methylene blue — do not induce divisions like azur, 
and yet they kill the cells by staining the chromatin 
of the nucleolus-centrosome. Why should only the 
latter dye cause cell-division; presumably both would 
cause death-struggles ? Moreover, we have often killed 
cells by prussic acid and nitro-benzol, but no division 
occurred and nothing resembling a death-struggle. 
Again, in connection with the experiment given in 
this chapter in which it was ascertained that the stage 
reached in a single act of mitosis varies directly with 
the quantity of the chemical substance absorbed, it 
appeared to us that if these mitotic figures induced in 
lymphocytes were in the nature of death-struggles, 
a cell once it had been started in its act of mitosis 
would continue that act until it was complete. But 
as the experiments showed, they did not do so, for 
when the diffusion of the chemical agent was arrested 
the mitosis ceased even in its early stages. 

These suggestions were worthy of consideration, 
and the only way to disprove them was to continue 
the investigations. It appeared to us reasonable to 
suppose that other cells, besides lymphocytes, would 
possibly respond by dividing to the chemical auxetic, 
and we also considered it possible that other chemical 
agents existed which would induce divisions. Lym- 
phocytes responded in such a constant manner, and 
always required a definite quantity of the substance 
that we thought it possible that there might be some- 
thing similar to it in the body which would cause their 
proliferation. The question was, What was this sub- 
stance and where was it contained ? Was it associated 



248 THE CYCLE OF CELL-DIVISION 

with the cell-proliferation of healing ? Lymphocytes 
proliferate in healing, especially in chronic healing, and 
chronic healing is a forerunner of cancer. Still 
leucocytes proliferate even more than lymphocytes in 
healing, but we had never up to this point, nor had 
any one else, ever seen a leucocyte divide. If the agent 
in the azur dye was analogous to a chemical substance 
in the body which caused the cell-proliferation of 
healing, it ought, strictly speaking, to cause the multi- 
plication of polymorphonuclear leucocytes as well as 
lymphocytes. But so far it had not done so. 

All these points were carefully considered at this 
stage, and they urged us to make further researches. 
Whether we were right or wrong in supposing that 
there might be a chemical auxetic in the body which 
caused proliferation of cells in a manner similar to 
the agent contained in the azur dye, we had made 
one step in causing one class of human cells to divide 
on the microscope stage by means of a chemical agent. 



CHAPTER XII 

THE "EXPERIMENTAL TEN MINUTES " DIVISIONS IN- 
DUCED IN THE SO-CALLED POLYMORPHONUCLEAR 

LEUCOCYTES METHOD FOR COUNTING THE NUMBER 

OF GRANULES CONTAINED IN EOSINOPHILE LEUCO- 
CYTES, AND THE REDUCTION OF THIS NUMBER IN 
THE CELLS OF CANCER PATIENTS. 

The reduced number of chromosomes exhibited in 
lymphocytes when they are forced to divide in ten 
minutes by increasing the diffusion of the stain into 
them with alkali reminded us forcibly that the cells 
were undergoing mitosis under the stress of experi- 
mental conditions. The somatic number of chromo- 
somes of human cells is 32 ; and since lymphocytes will, 
if their divisions are delayed, divide by the somatic 
number, it appeared to us that the normal time occu- 
pied by the division of the cells in the body is probably 
much longer than the ten minutes allowed to them 
on the microscope slide. It was appreciated that it 
would be better if we could delay the diffusion of the 
stain into the cells to such an extent as always to 
produce somatic divisions. But unfortunately herein 
we met with a difficulty which has not yet been over- 
come. As we have pointed out, death has been delayed 

249 



250 THE DIVISION OF LEUCOCYTES 

for twenty minutes while mitosis has been induced, but 
this experiment has only been followed by success on 
very few occasions. For general practical purposes it 
must be remembered that whatever is done in the way 
of attempting to induce divisions in cells when they are 
resting on a jelly-film, this must be done in ten minutes. 
If the diffusion of the chemical agent is delayed beyond 
this time, except in very few instances, the cells will 
refuse to divide at all, simply because they die. 

The reason for this is a question of vitality, which 
brings us back to the disadvantages of in-vitro experi- 
mentation. All cells of the body lose vitality gradually 
after they have been shed. White blood-corpuscles will 
live in citrate solution for two or three days at the room 
temperature, but they lose vitality all the time. As 
already pointed out, there is no known medium in 
which blood-cells will live and thrive, and in the best 
medium at our disposal they merely exist for this short 
period. When cells are resting on a jelly-film, how- 
ever, they are not even in the best available medium; 
but at present we are bound to employ the jelly 
method, for we have not succeeded in inducing divisions 
in any other way. The reason for this is two-fold: 
firstly, because substances can be made to diffuse into 
individual cells more quickly if the cells are pressed 
into the jelly which contains them; and, secondly, we 
think that lymphocytes prefer to be at rest when they 
divide, for we cannot induce divisions with the cells 
floating in a solution, although we have tried to do 
so many times in solutions which have contained the 
necessary constituents. 



EXPERIMENTAL TEX MINUTES 251 

At present there is nothing for it but to induce 
divisions with the cells spread out on jelly under a 
cover-glass; and it must be remembered throughout 
that these conditions are most detrimental to the cells. 
Pressed in this way into the jelly by means of a cover- 
glass, which to living cells must be proportionately of 
enormous weight, leucocytes and lymphocytes will not 
live more than about three-quarters of an hour. Al- 
though they will exist for this time, and although 
amoeboid movements may be excited in them during 
greater part of it, it is obvious that the cells are in 
reality dying slowly all the time. Since the ease with 
which one can induce divisions in lymphocytes varies 
directly with the vitality of the cells, it is clear that 
whatever is done to induce mitosis must be done 
quickly, and by practical experiment it has been found 
best to observe the general rule that, when one attempts 
to induce cells to divide on the microscope slide one 
must so arrange the jelly-film that the cells will be in 
the act of mitosis within ten minutes. This is a serious 
disadvantage appertaining to in-vitro experimentation, 
which cannot so far be overcome, and it is important 
to remember it throughout. The cells are labouring 
under abnormal difficulties which modify one's deduc- 
tions from the facts seen ; and since this important point 
will frequently have to be considered, it is convenient 
to standardise these detrimental conditions and desig- 
nate them the "experimental ten minutes." 

Two corollaries depend on the "experimental ten 
minutes." Since the induction of a division in a cell 
depends on the diffusion into it of a certain quantity of 



252 THE DIVISION OF LEUCOCYTES 

a chemical agent, and since the quantity of the agent 
required must increase with the rapidity at which we 
wish mitosis to occur, it is obvious that a greater con- 
centration of the chemical agent will be required to 
induce a division in the "experimental ten minutes" 
than would be required to make a cell reproduce itself 
if it were resting in its normal surroundings, where it 
might take a much longer time in its division. 

The second corollary is that if the jelly on which 
the cells are resting contains a saturated solution of a 
given substance which is diffusing into the cells to the 
utmost in ten minutes, and if that substance does not 
induce divisions in the "experimental ten minutes," it 
does not prove that that substance will not within the 
body, with the cells in their natural surroundings, cause 
them to proliferate. 

It was a matter of concern to us that the azur 
dye did not make the polymorphonuclear leucocytes 
(fig. 76) divide. So far only lymphocytes responded. 
If the contention was correct that the dye contains a 
specific agent which was possibly analogous to some 
similar agent in the body which causes proliferation 
of lymphocytes, it appeared reasonable to expect that 
some similar agent, if not an identical one, would also 
cause divisions in leucocytes; for the latter cells always 
proliferate together with, and to a greater extent than, 
lymphocytes during the process of healing. So far, 
however, we had not seen anything resembling a 
division in a polymorphonuclear leucocyte. It must 
be admitted that we had no idea as to what a leuco- 



SPECULATIONS REGARDING THEM 



253 




Fig. 76. — A resting polymorphonuclear leucocyte. Its granules are stained 
but not its nucleus. The cell was alive. 




Fig. 77. — A basophile leucocyte in the act of cell-division. The granules 
of the cell are in the centre. The lobes of the nucleus are at the poles of 
the cell which is dividing ito three. 



SPECULATIONS REGARDING THEM 255 

cyte would look like when it divided, for no one had 
ever seen a division in a leucocyte. These peculiar 
cells are large, and easily examined. They differ from 
all other cells in that they contain a polylobed 
nucleus, and it was very difficult to imagine how 
mitosis would occur in such a cell. Speculations have 
been made from time to time to the effect that these 
cells divide by pluripolar mitosis. Each lobe of the 
nucleus is said to undergo a mitosis of its own, and 
that the chromatin within the lobe forms up into 
chromosomes. This would mean that a cell with five 
lobes to its nucleus would divide into ten cells. Such 
a speculation makes no allowance for the Altmann's 
granules, which attain a large size in these cells, or 
for the filaments which unite the several lobes of the 
nucleus. Since the cytological process of mitosis in 
lymphocytes was so different to what was expected, 
we were prepared to see the speculation disproved; but 
in spite of this it must be admitted that when at last 
the divisions of leucocytes were seen the arrangement, 
of their cytological elements came rather as a reve- 
lation. 

Jelly-films were made which contained greater 
strengths of the azur dye, extracted from polychrome 
stain in the way which we have described. The 
possibility of divisions being induced in leucocytes 
was considered to be an event which would be seen 
before long; but when it was first seen it, like the 
first mitosis in lymphocytes, was not recognized or 
appreciated. The increased quantity of azur dye was 
added to the jelly in reality to see what the effect of 



%56 THE DIVISION OF LEUCOCYTES 

excess of it on lymphocytes might be. On one occa- 
sion a "basophile" leucocyte was found lying on the 
jelly with its granules arranged in rows, and forming 
a sort of radiating pattern. Moreover, the granules 
were in the centre of the cell, which is an unusual 
position for them, and there were clear spaces outside 
them which evidently contained the lobes of the nuclei, 
although the latter were not stained, as it is very diffi- 
cult to stain the nuclei of basophile leucocytes in vitro. 
The condition had not been seen before, but it was 
passed over, for at the time lymphocytes were being 
sought for. Some days afterwards another basophile 
cell was seen in a similar condition, and then it was 
more carefully observed. The lobes of the nucleus of 
this cell could just be made out, and they were external 
to the granules. The cell-wall itself was indented in 
three places, so that the leucocyte looked like the pro- 
peller of a steamship. The granules were deeply stained 
and turning black (fig. 89), which sometimes occurs in 
vitro in the stained granules of basophile cells ; and they 
were again arranged in indefinite lines or rows. It 
was this arrangement of the granules which specially 
arrested attention. The Altmann's granules of lympho- 
cytes form up into rows to form the chromosomes, and 
it looked as if something of some similar nature was 
happening on this occasion in a leucocyte. 

This curious condition of the basophile leucocyte 
seemed to have occurred in response to the excess 
of azur dye. Still more of it was therefore added to 
some coefficient jelly which also contained atropine, 
polychrome dye, and extract, with the idea of de- 



MITOSIS OF LEUCOCYTES 257 

liberately producing this condition of the basophile 
leucocytes. As a matter of fact, the jelly contained 
0.6 cc. of the azur dye, and its index of diffusion was 
now arranged for the coefficient of diffusion of the 
basophile leucocytes, which is the same as that of the 
ordinary neutrophile leucocyte. 

After removal from the incubator at the end of 
ten minutes, it was seen that the lobes of the nuclei 
of the neutrophile polymorphonuclear leucocytes were 
just staining a faint blue colour, and — there could be 
no question about it — nearly every leucocyte in the 
specimen was in the act of division. Neutrophile, 
basophile (fig. 77), and those eosinophile (fig. 78) 
leucocytes which were not ruptured were undergoing 
the act of reproduction on the jelly-film. They were 
dead owing to the staining of the lobes of their nuclei, 
but the lines of demarcation between the individual 
daughter cells could be distinctly seen. The cytological 
procedure by which these cells divide is identical in 
all varieties of leucocyte. As in lymphocytes, the 
Altmann's granules- were formed into rows, and pre- 
sumably they are analogous to chromosomes; the rows 
of granules become arranged into indefinite lines 
radiating outwards from the dividing-point, which is 
in the centre of the cell. Running down through the 
centre of the mass of granules, the filament which 
unites the lobes of the nuclei evidently forms a basis, 
analogous to the spindle of other cells, to which the 
chromosomes are attached; and at the poles of this 
filament, or spindle, the so-called lobes of the nuclei 
appeared. It was then immediately appreciated that 

J 7 



258 THE DIVISION OF LEUCOCYTES 

these bodies are in reality the centrosomes of the 
cells. 





If a leucocyte has two lobes to its nucleus it will 
divide into two cells; if it has three lobes it will divide 
into three cells, and so on. It will thus be seen that 
when these cells proliferate each daughter cell will have 
one centrosome until that centrosome itself divides and 
assumes the appearance of being polylobed. Further, 
a tissue made up of such daughter cells would be de- 
scribed as consisting of "mononuclear cells." The 
chromatin-staining lobes within the leucocytes are there- 
fore not nuclei but centrosomes, and the so-called 
Altmann's granules, which have been variously sur- 
mised to be collections of food or secretion, are the 
elements of the chromosomes themselves. 1 As in 
lymphocytes so in leucocytes, the chromosomes are 
outside the nucleus. Divisions have been induced in 
hundreds of leucocytes, and the procedure is always 
the same in all of them (figs. 79-86). 

Now, the increased quantity of the azur dye con- 
tained in the jelly did not improve the mitosis induced 
in the lymphocytes; in fact, it seemed too strong for 

1 Professor Sherrington has a specimen of an eosinophile leucocyte of a 
cat in which the individual granules are elongated and almost rod-shaped. 
We have also seen elongated granules in these cells in human blood. 



MITOSIS OF LEUCOCYTES 



259 






Fig. 78. — An eosinopnile leucuc,y ie m uie eaiiiesi stage oi division. The 
granules are arranged in lines radiating outwards from the centre of the 
cell. The lobes of the nucleus were at the poles. 






Fig. 79. — Early stage of division ot a neutrophile leucocyte. 



MITOSIS OF LEUCOCYTES 



261 





Fig. 80. — A dividing leucocyte. 




Fig. 81. — A dividing leucocyte. 



MITOSIS OF LEUCOCYTES 



263 






Fig. 81a. — Division of a leucocyte. The linear arrangement of the granules 
could be well seen. 




Fig. 82. — A dividing leucocyte. 



MITOSIS OF LEUCOCYTES 265 




Fig. 83. — A dividing leucocyte. 






Fig. 84. — A dividing leucocyte. 



MITOSIS OF LEUCOCYTES 



mi 




^ 




Fig. 85. — A dividing leucocvte. 



' 



...„ ': '-- * a.- * . 





Fig. 86. — A dividing leucocyte. 



MITOSIS OF LEUCOCYTES 269 

them, as only early stages of mitosis were seen in 
them. Hence it became apparent that the auxetic 
constituent of the aniline dye which induces divisions 
in lymphocytes also does the same thing with leucocytes ; 
but it evidently requires more of it, ceteris paribus, 
to induce a division in a leucocyte than in a lymphocyte. 
The coefficient of diffusion of the lymphocyte is higher 
than that of the leucocyte if the staining of the nucleus 
is the moment by which it is determined; but so far 
as inducing divisions is concerned the coefficient of 
lymphocytes seems to be lower, for they require less 
of the chemical agent than do leucocytes. 

The divisions of reproduction had now been induced 
in both leucocytes and lymphocytes by an artificial 
chemical agent. These cells are the ones which pro- 
liferate when a tissue is damaged, and it is by their 
multiplication that the healing of an injury takes place, 
and it must be borne in mind that cancer, with its in- 
creased malignant proliferation, is intimately associated 
with chronic healing. Judging by the divisions induced 
in these white blood-corpuscles it appeared that their 
reproduction takes place in a cycle which depends on 
some chemical substance absorbed by them. The cycle 
consists apparently of the division of the centrosomes, 
division of the chromosomes, and the division of the 
cell. Whether there is a "resting stage" in the strict 
sense of the term, we are not in a position to state, 
for we do not know how long a time is occupied in 
the division of the centrosome. If a cell is absorbing 
the agent which causes it to divide, presumably the 
cycle of mitosis is going on in direct proportion to the 



270 THE DIVISION OF LEUCOCYTES 

amount of the agent absorbed. The division of 
the centrosome seems to be part of this cycle, but 
how long this part takes we do not know. Hence it 
appears possible that what is commonly known as 
the resting stage is in reality the time occupied by 
the division of the centrosome. 

The method of division of leucocytes and lympho- 
cytes is so constant that we thought it was reasonable 
to expect that the proliferation of healing would be 
ultimately proved to take place by a similar process, 
and that if so there must be produced in an injured 
tissue some chemical substance very similar in its effects 
to that contained in azur dye. Up to this time, however, 
we had not succeeded in inducing divisions at all with 
any substance which we could call a "natural" sub- 
stance. There is nothing in the body that we know of 
at all like the aniline dye. It was true that an extract 
of dead haemal gland augmented the action of the ani- 
line dye; but it would not induce divisions by itself. 
Extracts of tissues other than haemal gland were tried, 
made in the same strength — namely, 100 per cent — and 
it was found that suprarenal glands of sheep augmented 
the action of the stain in inducing divisions even better 
than haemal gland, and several extracts, such as those of 
muscle and liver, did the same, but to a lesser degree. 
In spite of the augmenting action of all these extracts, 
however, none of them alone in the strength tried would 
induce divisions either in lymphocytes or leucocytes in 
the experimental ten minutes. 

This inability to cause cell-division by entirely 
"natural" substances, such as the extracts named, was 



THE EXTRACTS CONCENTRATED 271. 

believed, after mature consideration, to be due to the 
fact that we had not tried extracts in sufficient strength 
for cells to respond to them under the detrimental 
circumstances of the "experimental ten minutes. " It 
has already been pointed out as a corollary to these 
circumstances that if a cell refuses to respond to a given 
substance by not dividing in the experimental ten 
minutes, it does not prove that that substance does 
not actually contain an active principle for inducing 
cell -division. We therefore considered the advisability 
of concentrating the extracts, and then trying them 
again by themselves. 

In the first instance this concentration process took 
some little time. At this stage of our researches we 
were unaware that the active augmenting principle 
contained in the extracts was " thermostable " and 
would resist boiling, and in consequence, at the outset, 
we evaporated the extracts at the room tempera- 
ture, which was a most tedious process. Moreover, 
during this slow concentration of the extracts it was 
necessary to test them from time to time to see if their 
augmenting action was impaired at all with keeping. 

In the meantime other points were considered. It is 
well known that cancer-cells frequently are seen to be 
dividing with a reduced number of chromosomes. As 
we have already stated, we believe that a reduction in 
the number of chromosomes is due to increased pro- 
lificity in cells; and this being the case, it seemed 
probable that there might be some increase in or 
augmentation of the cause of proliferation of the cells 
of cancerous growths. Further, if this is the case, the 



272 THE DIVISION OF LEUCOCYTES 

augmenting substance might appear in the peripheral 
circulation. It has already been pointed out in 
Chapter VIII. that cancer plasma excites amoeboid 
movements in leucocytes, and that alkaloids also excite 
these movements. Since the alkaloid atropine augments 
the action of azur stain in inducing divisions, it was 
thought possible that the exciter of amoeboid move- 
ments found in cancer plasma might be in the nature 
of an alkaloid possibly derived from the neighbourhood 
of the growth. Now, atropine and stain together cause 
white blood-cells to extrude granules of chromatin, a 
phenomenon which we erroneously called "flagellation" 
(see Chapter X.), and this extrusion had also been 
observed in cells which have been subjected to cancerous 
plasma. 1 The suggestion followed that the cells might 
be extruding their granules deliberately in response, not 
only the artificial combination of stain and alkaloid, 
but also to some possibly similar combination derived 
from the malignant growth. Moreover, since in both 
the artificial and the natural circumstances, cells appear 
to divide with a reduced number of chromosomes, and 
since the granules form the chromosomes, it was sur- 
mised that the extrusion might be part of the process 
of reduction. It must be remembered that our ex- 
perimentation left us convinced that the divisions of 
lymphocytes and leucocytes occur just as the stain is 
combining with the chromosome-granules; and as the 
extrusion of the granules — which has been seen by 
others as well as ourselves — seems to be a deliberate 

1 "The Flagellation of Lymphocytes in the Presence of Excitants both 
Artificial and Cancerous," by H. C. Ross and C. J. Macalister, British 
Medical Journal, January 16, 1909. 



COUNTING GRANULES SUGGESTED 273 

action on the part of the cells, tve went so far as to 
theorise that the cells might be discarding their granules 
in order to prevent some' of the combination of the 
"auxetic" and their granules, and so delay their pro- 
liferation to some extent. 

This theory led to the suggestion that we should 
try to count the number of granules contained in the 
blood-cells of cancer patients, with a view to see if they 
were reduced in number in that disease. The blood of 
cancer patients seems to contain a body which excites 
amoeboid movements and the extrusion of granules, 
and, therefore, the blood-cells themselves as well as the 
cells composing the growth might also have a reduced 
number of the granules which form their chromosomes. 

We must admit that these suggestions were based 
on slender grounds of evidence, and it was appreciated 
that to count the number of granules of the leucocytes 
of cancer patients would require considerable work, 
especially as many control experiments would have to 
be made, for we did not even know the normal number 
of granules in healthy persons' cells. Still, it was very 
necessary to try to find out whether the clue on 
which we were engaged was in any way correct, and 
it was realised that in order to make the counts it 
would be necessary to examine a large number of 
samples of blood-cells from many patients and from 
normal and other persons- — a procedure which had not 
yet been done by this in-viiro method. Hence no 
matter how far-fetched it appeared at first sight for us 
to count the granules of blood-cells in cancer patients, 
I thought that an endeavour to do so would be justified, 
and I devised the following technique for doing so. 

18 



274 THE DIVISION OF LEUCOCYTES 

It was seen from the outset that it would be quite 
impossible to count the number of granules contained 
in lymphocytes, and the same could be said of those 
of the common neutrophile leucocyte (fig. 76). But it 
is possible to do so in the so-called eosinophile cells 
(fig. 87). These cells have large granules, which stain 
a deep scarlet with the polychrome dye, and therefore 
these cells were chosen for this series of experiments, 
especially as they are fairly common (2 to 4 per cent) . 

Three difficulties presented themselves in arranging 
a technique for counting the number of eosinophile 
granules : 

1. To the novice the basophile cell is sometimes 
very difficult to distinguish by in-vitro staining from 
the eosinophile cell, and mistakes seriously modify the 
results. If specimens of each class of cell are seen 
lying side by side (fig. 88) there is no difficulty in 
distinguishing them, the eosinophile cell being much 
the larger, although there is very little difference be- 
tween the size of their granules. But in spite of the 
fact that the cells rarely are thus found lying side by 
side, with a little experience they can be readily dis- 
tinguished; the granules of the basophile cell are more 
discrete, and the lobes of its nucleus will practically 
never stain by this in-vitro method. 

2. A living leucocyte is spherical in shape, and it 
usually appears with its granules heaped one on top 
of another, rendering it impossible to count them 
accurately. 

3. If one attempts to count through the microscope 
a group of granules not arranged in any definite order, 



COUNTING GRANULES 



275 



Fig. 87. — An eosinophile leucocyte with its granules stained. 



COUNTING GRANULES 277 

one is apt to count the same granule more than once, 
and it is eas} 7 to lose one's place — in which case it 
becomes necessary to begin all over again. On looking 
at the cells through the microscope, the granules appear 
as though they might with care be counted, and it is 
most inviting to attempt to do this and to rely on it; 
but on testing this rough-and-ready method we have 
found that it usually involves an error of nearly 50 per 
cent. No estimate whatever can be made of the 
number of granules contained in a cell by merely 
looking at it through the microscope, no matter what 
magnification is used. 

Obviously the granules must be stained, and then 
it is necessary: (1) to distinguish readily between an 
eosinophile and a basophile leucocyte; (2) to kill the 
cells, and then to burst them so as to cause their 
stained granules to rest discretely side by side in one 
plane and not on top of one another; (3) to magnify 
the image of the ruptured cell in such a way that one 
can "tick off" each granule with a pencil on paper 
as it is counted, so as to avoid counting the same 
granule twice over. 

By the following procedure the staining, killing, 
differentiation, and bursting can be readily accom- 
plished. In order to magnify the image of the ruptured 
cell so as to count its granules and to "tick them off," 
it is necessary to obtain a photomicrograph negative of 
it, and then to project the photographed image on to 
a paper screen with an optical lantern, when the image 
of each granule can be marked off on the paper with 
a pencil. 



278 THE DIVISION OF LEUCOCYTES 

It is necessary to employ the photomicrographic 
apparatus which I have already described, and the 
photographs must be taken with as little delay as 
possible after the cells have been ruptured. Unfixed 
cells may rapidly become achromatic after death, and, 
in the case of a ruptured cell, the loss of stain may 
occur with great rapidity. 

The blood of the person to be examined is drawn 
into a capillary tube and there mixed with an equal 
volume of citrate solution. At the room tempera- 
ture this solution will keep the cells alive for some 
days; but when it is intended to count the granules of 
the eosinophile leucocytes, it is better to examine the 
blood as fresh as possible. 

A jelly is prepared thus: To a tube containing 
5 cc. of coefficient jelly add 4 units of Unna's poly- 
chrome stain, 7 units of the 5-per-cent alkali solution, 
and, instead of making the contents of the tube up 
to a total of 10 cc. with water, 3.9 cc. of a molten 
2-per-cent solution of agar in water is used. The 
last solution contains agar in order to make the jelly 
exceptionally firm, so that the ultimate bursting of the 
cells can be facilitated. The jelly is melted and boiled 
and a drop of it run on to a slide, where it is allowed 
to set. A drop of the citrated blood is then placed 
on a cover-glass, which is inverted and allowed to fall 
flat on the film in the usual way. The slide is then 
placed in the 37° C. incubator for three minutes exactly. 
When examined microscopically it should be seen that 
the nuclei of the eosinophile leucocytes are just staining 
scarlet, showing that death is occurring; the granules 



COUNTING GRANULES 



279 




Fig. 88. — A tieia containing a neiurophile, an eosinophile, and a baso- 
phile leucocyte. The upper cell is the neutrophile and the lower one the 
basophile cell. All the cells are ruptured, but their granules are stained. 




Fig. 89. — A basopniie leucocyte wnose stained granules have been turned 

black by heat. 



COUNTING GRANULES 281 

of the cells should be deeply stained. If the nuclei 
are not yet stained, a little more alkali must be added 
to the jelly and a fresh specimen made. If the cells 
are achromatic or disorganised, or if the nuclei of the 
neutrophile cells are deeply stained, the jelly is too 
alkaline, and a little acid solution must be added to it. 
But if the coefficient jelly and other solutions are 
correct, the nuclei of the eosinophile cells will just be 
staining. 

Using a J-inch or equivalent objective the specimen 
is searched until a suitable eosinophile cell is found. 
If a cell is distorted or hemmed in by red cells, it is 
necessary to pass it over and find another. 

If there is any doubt as to whether a cell is an 
eosinophile or basophile one, the slide is removed from 
the mechanical stage in such a way that on returning it 
to the microscope the same field can be focused again. 
The slide is then again incubated for three minutes, but 
at 47° C. On examination of the cell, if it is an eosino- 
phile leucocyte, its granules will still appear scarlet ; but 
if it is a basophile cell, its stained chromosome granules 
will have turned black 1 (fig. 89). With a little experi- 
ence of the method of staining, however, the difference 
between the classes of cell can be detected without this 
procedure of incubation at 47° C, which is apt to cause 
premature rupture and achromasia. 

The next step is to burst the cell. The photo- 
micrographic apparatus being ready on its slide above 
the observer's head the immersion objective is " turned 

1 These granules may turn black at 37° C. We have no explanation to 
offer of this phenomenon. 



282 THE DIVISION OF LEUCOCYTES 

on" and focused, and the cell is brought into the 
centre of the field. Watching it through the eye- 
piece, keeping one hand on the fine adjustment, 
the cover-glass, which of course is resting on the 
jelly-film, is gently struck (tapped) with a glass rod 
held in the other hand. At each tap the cells are 
seen to be jerked out of the field, but, provided the 
taps are not too forcible, the eosinophile cell can 
easily be followed by using the mechanical stage. 
It is usually necessary to strike the cover-glass two 
or three times, and generally at the third blow the 
eosinophile is seen to totter and then burst, scattering 
its stained granules about on the surface of the jelly 
in the field of the microscope. This is a trick, of 
course, which was devised by one of us (J. W. C), 
and with a little practice rupture can nearly always 
be assured. 

When a cell ruptures on this jelly — which contains 
salts — its nucleus loses its stain instantly, but at the 
room temperature the granules do not usually become 
achromatic for some little time. On the other hand, 
in some instances they may become unstained in a 
few moments, and for this reason, in order to secure 
the photographic negative, speed is now required. 
The ruptured cell is placed in the centre of the field 
with the mechanical stage; the working eye-piece is 
removed from the microscope, the camera is allowed 
to slide down the wooden slide, and its projecting 
eye-piece, which is already attached to it by means of 
a flexible velvet collar, is inserted into the draw-tube 
of the microscope. By the simple movement of swing- 



COUNTING GRANULES 283 

ing the microscope mirror on its gimbals out of the 
focal axis, the working 32-c.p. gas-light is changed 
to the water-cooled ray of light from the 1-amp. 
Nernst lamp. The image of the ruptured cell will 
then be seen on the ground-glass screen at the back 
of the camera, where it can be rapidly focused. 

The special precautions regarding the focusing 
with this method have already been described, but 
it should be remembered that in order to be able to 
count the number of the granules in the ruptured 
cell it is most important to obtain as perfect a nega- 
tive (figs. 90, 91) as possible. 

If the photography has been accomplished quickly, 
the camera may be pushed up out of the way, the 
microscope mirror replaced, and the specimen may be 
searched for more eosinophile leucocytes. 

To count the number of granules contained in a 
ruptured cell, the negative must — after it has been 
developed and dried in the usual way — be placed in an 
optical lantern, and the image of the ruptured leucocyte 
projected on to a screen which has a sheet of white 
paper pinned in front of it. One stands close in front 
of the screen and counts the granules, each of which 
will now appear about the size of a shilling-piece, and 
the image of each granule can be "ticked off" with 
a pencil on the paper (figs. 92, 93) . It is thus impossible 
to count any granule twice over, and an accurate 
enumeration can be made. 

Such is the technique. By it there have been 
counted 38,759 granules from 235 cells from 96 persons, 



284 THE DIVISION OF LEUCOCYTES 

22 of whom were suffering from undoubted 1 cancer, 2 
and 47 of whom apparently were not. Of the latter, 
which for convenience will be called the " control" cases, 
some were "healthy" and others were suffering from 
various diseases (hospital patients) . We did not count 
the granules in each of the 235 negatives as the latter 
were obtained, but the plates were developed and then 
put away until a hundred or more had collected. The 
name of the person from whom the blood had been 
taken was entered into a book with the age, sex, 
disease (if any), and other details. The negatives were 
numbered consecutively, and the numbers corresponded 
with similar ones in the book against the names of the 
persons from whom the cells had been derived. The 
samples of blood were taken from persons, cancerous or 
otherwise, as they came into hospital, and therefore, 
without referring to the book, a number on a negative 
gave no indication from whom the cell it depicted was 
derived. With three exceptions, the samples of blood 
were collected and photographed by one of us, who 
kept the book in his laboratory. The counting was 
done by another, who had no idea to whom the 
numbers on the negatives referred. 

The only possible source of error is in the counting. 
Some of the negatives were not quite perfect, and 
some of the granules appeared blurred ; hence there may 
be a small error in some of the numbers, but it cannot 
be very important judging by the uniformity of the 
averages. 

1 Determined either by such clinical manifestations as recurrence or 
metastasis, or by pathological examinations. 2 Carcinoma. 



THE NUMBER OF GRANULES 



285 




Fig. 90. — One ot the negatives of a ruptured eosinophile leucocyte (negative 

No. 52). 




Fig. 91. — One of the negatives of a ruptured eosinophile leucocyte (negative 

No. 54). 



THE NUMBER OF GRANULES 287 




Fig. 92. — Counting the granules. The image of the ruptured cell 
depicted on negative No. 52 is projected on to a sheet of white paper pinned 
on to a screen. 




Fig. 93. — Counting the granules of negative No. 54. 



THE NUMBER OF GRANULES 289 

At first only two cells were taken from each person, 
but since it was found that there was frequently a wide 
variation in the number of granules contained in in- 
dividual cells, this number was afterwards increased to 
five. Averages were then struck, and the tables given 
in Appendix I. give the number of leucocytes ex- 
amined and the persons from whom they were derived, 
together with the number of granules contained in the 
largest and smallest cells from each person. From 
these averages it will be seen that the sex makes 
practically no difference in the average number of 
granules contained in the cells; but more experiments 
will be needed before the same can be said about age. 
The averages can, in the first place, be divided into 
two groups, male and female. Each of these groups 
can be subdivided into two, viz. control persons 
(healthy and diseases other than cancer), and cancer 
persons. Neglecting fractions, the average number of 
granules in the cells appear thus : 

Number of Average number of granules in cells of 

persons males females 

47 controls 168 .. . 168 

22 cancer 159 .. . 161 

Thus, between normal 1 males and females there is no 
difference, and between carcinoma males and females 
there is very little difference; but the number of 
granules in cancer-cells is well below the normal in 
the averages of both males and females, and it would 
appear from this that the number of granules con- 
tained in the cells of cancer patients is actually reduced. 

1 Control cases (normal, and diseases other than cancer) . 

i9 



290 THE DIVISION OF LEUCOCYTES 

The table and its summary supply further details. 
As was expected, the reduction is not very large, but 
the striking point is that, in addition to the total 
cancer averages being below the normal, a subdivision 
into such groups as male and female demonstrates 
that the reduction in cancer is again present in both 
groups. 

Every individual case of cancer in the category 
does not, by any means, have a reduction in the 
average number of granules contained in the cells, and 
it will be seen that many of the individual controls 
showed a reduction; but when one comes to deal 
with comparatively large numbers, the reduction in 
carcinoma is demonstrated. It must be remembered 
that in everybody there is a great variation in the 
actual number of granules contained in individual cells ; 
and when sampling say five cells from a person, one 
may by chance hit upon five larger or five smaller 
cells. Obviously, therefore, it is only by the observa- 
tion of many cells from large numbers of persons that 
one can reduce to a minimum the "error of random 
sampling." We think, however, that the enumeration 
of the granules contained in 235 cells, from 22 cancer 
patients and 47 controls, diminishes this error to such 
an extent that the results are fairly trustworthy. At 
the same time, it must be remembered that in experi- 
mentation of this nature the error of random sampling 
can never be altogether eliminated, and therefore the 
reliability of the averages depends entirely on the 
extent of this error among the cells which have been 
photographed. 



THE NUMBER OF GRANULES 291 

Among the control cases three cases of sarcoma 
are included. In all of them there was no apparent 
reduction, and the same can be said of another case 
tested recently. But the number of cells is too small 
to form any conclusion from, and more cases will be 
required. 



CHAPTER XIII 

THE AUXETIC ACTION OF CANCER SERUM THE IN- 
DUCED DIVISIONS OF GRANULAR RED CELLS 

THE AUXETIC ACTION OF " THE REMAINS OF 
DEAD TISSUES," AND ITS AUGMENTATION BY 
ATROPINE AND THE PRODUCTS OF PUTREFACTION 

THE ISOLATION OF THE AUXETICS KREATIN 

AND XANTHIN DISCOVERY OF THE CAUSE OF 

THE CELL-PROLIFERATION OF HEALING 

Counting the granules of eosinophile leucocytes from 
cancer patients, therefore, seemed to us to show that 
the clue on which we were working was to some extent 
correct. Judging from the comparison between the 
number of granules contained in the cells of cancer 
patients and those of other people, there appears to 
be a reduction in cancer, and this reduction presumably 
is due to the presence in the blood of some agent which 
causes more proliferation than normal. It has been 
pointed out that increased prolificity owing to excessive 
absorption of a chemical agent makes cells divide by a 
reduced number of chromosomes as seen in carcinoma 
cells ; and now apparently other cells, such as the eosino- 
phile leucocytes, have in that disease a slightly reduced 

292 



AUXETIC IN CANCER PLASMA 293 

number of chromosome granules. But this digression 
from the main researches also taught us that other 
facts were to be learnt from the comparison of samples 
of peripheral blood from twenty-two cancer patients 
and forty-seven " others." Never before had systematic 
examination of blood from such groups of persons been 
made by the in-vitro staining of their cells, and it was 
soon noticed that in the samples of cancer blood the 
actual number of eosinophile leucocytes was reduced; 
in fact, four cases could not be included in our category, 
because, even after repeated examination of many 
samples of their blood, no eosinophile leucocytes could 
be found; and in all the other cases, with the exception 
of three, there was an undoubted reduction in the 
number of eosinophile cells. In the three exceptions 
there appeared to be an eosinophilia. 

In some cases of carcinoma, also, there was a large- 
lymphocytosis, especially in the advanced cases. But 
this is by no means an absolute rule, and, moreover, a 
large-lymphocytosis was fairly common among the 
control specimens. 

But a still more important point was observed. 
We have already shown how a mixture of azur dye 
and atropine causes excitation of amoeboid movements 
in leucocytes and lymphocytes, then the discard of 
granules (flagellation), and lastly augmented cell- 
division; also that an agent has been detected in 
the plasma of carcinoma patients which induces the 
first two — i.e. excitation of amoeboid movements 
and the discard of granules. We have just shown 
that the granules in certain leucocytes in cancer 



294 THE CAUSE OF HEALING 

patients are reduced in number. The inference is that 
this reduction is made in response to the same agent 
which causes the excitation and discard of granules. 
The important point is that while engaged in these 
blood examinations the fact became apparent that this 
agent in cancer plasma (presumably it is the same 
agent) will help to induce cell-division. 

The large lymphocyte requires a considerable 
quantity of stain, extract, or atropine before it will be 
induced to divide in the "experimental ten minutes." 
In the technique, described in the last chapter, for 
counting the granules of eosinophile leucocytes the 
jelly employed contains only 4 units of polychrome 
dye, the efficiency of which for inducing divisions is 
infinitesimal (the jelly containing no extract of dead 
tissues or atropine). Yet in the examination of the 
blood of three of the cases of carcinoma some of the 
large lymphocytes showed well-marked stages of early 
mitosis, whereas this result could not be obtained in 
any of the controls. It is clear, therefore, that the 
cells in these three cases were inclined to divide before 
they were ever placed on the jelly, and the trifling 
assistance which they received from the 4 units of the 
polychrome dye caused them to show well-marked 
mitotic figures (figs. 62, 64), whereas the large lympho- 
cytes in all the control specimens, made under exactly 
the same conditions, remained at rest. 

Moreover, in two other cancer patients (both cancer 
of the stomach), owing to anaemia, many granular red 
cells were seen in their blood. On its being examined 
on jelly which contained azur dye, extract, and atropine, 



CONCENTRATION OF EXTRACTS 



295 




Fig. 94. — A dividing red cell from a cancer patient. 




Fig. 95. — A dividing red cell from a cancer patient. The granules seem 
to be arranged in an indefinite figure. 



CONCENTRATION OF EXTRACTS 297 

amitotic 1 divisions (figs. 94, 95) were induced in these 
granular red cells. The granular red cells of normal 
and other persons have never hitherto been seen to 
make any attempt to divide on auxetic jelly or any 
jelly, and hence it appears that these cells from these 
cancer patients are also more prone to divide than 
those of other people. 

Since it has been shown that the reproduction, 
certainly of lymphocytes and leucocytes, and possibly 
of other cells, depends (on the microscope slide) on 
the quantity of an auxetic absorbed by them, it is 
reasonable to suggest that the plasma of these cancer 
patients contained some such agent which caused this 
inclination to divide on the part of the large lymphocytes 
and red cells. Presumably this is the same agent 
which had been previously found to cause excitation 
of amoeboid movements, and the discard of granules 
for the combination of stain and atropine will also 
do this as well as cause augmented divisions. 

It is interesting to note that it is only the red 
cells which have granules which can be induced to 
divide, for it bears out the theory that the auxetic 
contains a specific agent which induces cell-division 
by acting on cell-granules. 

We may now return to the study of the extracts. 
It may be remembered that we had only succeeded 
in inducing divisions in lymphocytes and leucocytes 
with the artificial azur dve. Extracts of several 



1 We are uncertain whether some of the granular red cells were not 
dividing mitotically (fig. 95), as their granules appeared to be arranged in an 
indefinite figure. 



298 THE CAUSE OF HEALING 

dead tissues, especially that of suprarenal gland, in 
the strength of 100 per cent, would augment the action 
of the azur dye, but they would not in themselves 
induce divisions or even the early stages of mitosis 
in the experimental ten minutes. We had therefore 
made arrangements to concentrate these extracts so 
as to see if they would, if used in greater strength, 
induce divisions by themselves. At first it was thought 
better not to boil down the extracts for fear that the 
boiling might spoil the substance which augmented 
the action of the dye. The extracts were therefore 
placed in test-tubes, which were lightly plugged and 
put aside in the laboratory. As already mentioned, 
it was necessary to test these extracts from time to 
time to see whether they might become more effective 
as concentration occurred. When they were originally 
made they were sterile, because it may be remembered 
that they had been kept at 60° C. for twelve hours after 
filtration. Repeated examination of some of the tubes, 
however, caused them to become infected, and in 
consequence putrefaction set in in those tubes. After 
they had all been kept for three weeks it was noticed 
that the augmenting action of the contents of one 
of the infected tubes of suprarenal extract seemed 
to be increased. One cc, or even a few drops, of 
this extract, if added to the azur dye and made up 
in a jelly, caused advanced mitosis in lymphocytes, 
whereas with the other sterile tubes it seemed to require 
about the usual quantity of extract to augment the 
action of the dye. It was particularly noticed that 
this tube which contained so efficient an extract had 



DECOMPOSITION OF EXTRACTS 299 

been examined on several occasions, and owing to the 
infection of its contents the latter was in a foul-smelling 
condition. The increased augmentation when this 
decomposed extract was used was so remarkable that 
we decided to try its action by itself without any azur 
or other stain. 

The jelly was made up thus: To 5 cc. of coefficient 
jelly 3 cc. of the putrid extract, and 0.8 cc. of 
5-per-cent solution of sodium bicarbonate (8 units of 
alkali) were added. The alkali was present in order 
to cause the contents of the jelly to diffuse into the 
cells. The jelly was made up to a total of 10 cc. with 
1 . 2 cc. of water. In order to prevent coagulation of the 
extract a film was prepared from the jelly in the 
following way: The coefficient jelly was melted and 
boiled, and it was only as it cooled that the extract 
was added, the film being made immediately before 
the jelly had set in the test-tube. Fresh blood from 
the finger was spread on the jelly in the usual manner 
under a cover-glass. After incubation for ten minutes, 
an examination showed that some of the lymphocytes 
appeared to be in an early stage of mitosis. Now, 
we could not be very certain about this point, because 
no stain was present and consequently the chromo- 
somes were unstained and almost invisible. If mitotic 
divisions are sometimes difficult to see in stained 
specimens, they are much more difficult to distinguish 
when no stain is employed. Still, the cells looked 
rather as if they were attempting to divide (fig. 96). 

A fresh jelly was made, but it contained 1 cc. of 
alkali solution instead of 0.8 cc. ; and now there was 



300 THE CAUSE OF HEALING 

no doubt about it — this extract did actually induce 
mitotic figures in lymphocytes in the experimental 
ten minutes (figs. 97, 98). No azur stain, atropine, or 
other "augmenter" was added; the decomposed 
suprarenal extract induced mitosis by itself. 

Of course we thought at first that this result 
was due to the concentration of the extract; but 
this thought was soon dispelled by trying some of 
the other tubes which had been kept alongside of 
the ones which had so often been examined, the 
contents of which had decomposed. The sterile 
extract contained in these tubes would not induce 
divisions by themselves. Moreover, at the temperature 
of the laboratory, since they were kept in plugged 
tubes, the extracts did not evaporate very fast, and 
it was appreciated that they could not be so very 
concentrated. Some of the effective putrid extract, 
therefore, had water added to it, so that it was again 
made up to its original strength of 100 per cent. It 
was then made up in a jelly as before, and to our 
astonishment again it induced divisions in lympho- 
cytes; and what is more important, it induced the 
asymmetrical one-sided mitosis in many instances 
(fig. 99). 

A series of control experiments was then made. 
Jellies which contained only the salts sodium citrate, 
sodium chloride, and the 1 cc. of alkali were first 
tried, and no divisions could be seen. Then yet 
another series of experiments with fresh extract of 
suprarenal gland was made, once more without re- 
sult, and so it was ultimately proved that it was 



AUXETIC IN EXTRACTS 



301 




Fig. 96. — Very early stage ot mitosis in a iympnocyte induced by decom- 
posed extract of suprarenal gland. No stain. 




■HHBH 





Fig. 97. — Mitosis of a lymphocyte induced oy uecomposed suprarenal 
extract. No stain. 



AUXETIC IN EXTRACTS 303 





Fig. 98. — Mitosis induced in a lymphocyte by decomposed extract. No 

stain. 






Fig. 99. — Asymmetrical division induced by decomposed extract. No 
stain or atropine is present. 



AUXETIC IN EXTRACTS 305 

unquestionably due to the putrefaction that this one 
tube of extract induced divisions in the experimental 
ten minutes. 

Now, this fact required very careful consideration. 
A 100-per-cent solution of extract would not in itself 
induce divisions in lymphocytes unless it was putrid. 
When it is fresh this extract is not effective in the 
experimental ten minutes. It appeared probable that 
the extract does in itself contain some substance 
which causes cell-division, but in the strength of the 
extract of 100 per cent this substance is not present 
in sufficient quantity for it to induce divisions in the 
experimental ten minutes unless the whole extract is 
putrid. The first thing to do was to concentrate 
the extract and see if this theory was right. It was 
appreciated that the concentration process at the 
room temperature was a most unsatisfactory pro- 
cedure, for if the extracts were tightly plugged 
they did not evaporate down, but if they were left 
open they became putrid. One of the jellies which 
induced divisions by virtue of the putrid extract was 
therefore boiled and tried again. Still it induced 
divisions in lymphocytes. It was submitted to pro- 
longed boiling, and yet it was effective. So it was 
proved that the substance which it contained which 
caused cell-division was thermostable. We can 
boil these extracts with impunity, and their auxetic 
action is not impaired. Hence we made some fresh 
extract of suprarenal gland and evaporated it down 
to dryness by boiling. It is, when dry, a hygroscopic 
brown mass which is readily soluble in water. One 



306 THE CAUSE OF HEALING 

hundred grammes of sheep's suprarenal glands yields 
about 4 grammes of dry extract. 

A series of jellies were prepared which contained 
. 8 cc. of alkali solution (8 units) , variable quantities of 
solutions of the extract, and they were always made up 
to the total of 10 cc. with water. At first a 5-per-cent 
solution of the extract was made ; and it was found that 
if the jelly contained 1 cc. of this extract, very early 
divisions can be induced in lymphocytes. With 2 cc. 
later stages of mitosis will appear (figs. 100, 101); and 
if instead of the 5-per-cent solution a 10-per-cent one 
is made, even more marked divisions can be induced 
by this fresh extract alone in the experimental ten 
minutes. The best jelly to make in order to cause 
suprarenal extract to induce divisions in lymphocytes 
in the ten minutes is: 5 cc. of coefficient jelly, 1 cc. of 
alkali solution, 2 cc. of a 10-per-cent solution of dried 
suprarenal extract, and 2 cc. of water. By means of 
this jelly advanced mitotic figures can be induced in 
lymphocytes. 

So it was proved, therefore, that this extract of dead 
suprarenal gland contains a substance which will cause 
the divisions of lymphocytes. A fresh jelly was then 
prepared the same as the last one, except that it had 
added to it four more units of alkali solution. Now, as 
we anticipated, the polynuclear leucocytes also divided 
on the microscope slide (fig. 102). 

But the question was then asked, How was it that 
the original extract, although it was not strong enough 
to induce divisions by itself in ten minutes, did become 
effective when it was decomposed by putrefaction ? It 



AUGMENTED BY PUTRIFICATION 



307 




L 



Fig. 100. — Mitosis induced by fresh extract of suprarenal gland. No 
stain or augmentor present. 




• 






* 








Fig. 101. — Mitosis induced by fresh suprarenal extract. No stain is present. 



AUGMENTED BY PUTREFACTION 309 

was evident that the first extracts which we tried were 
not strong* enough to induce divisions in the ex- 
perimental ten minutes. If they became putrid, 
however, they apparently were. 1 The putrid solution 
was again tried, and again the asymmetrical divisions 
were seen. Now, these asymmetrical divisions are 
frequently induced by azur dye when it is augmented 
by atropine, and therefore we thought that it might 
be possible that the putrefaction of the extract might 
produce in it an augmenting substance which acted 
like the atropine. 

Fresh suprarenal extract was then made, and after 
it had been dried it was redissolved in water. It was 
made up in a 10-per-cent solution, and various quanti- 
ties of it were added to jellies which contained 1 cc. 
of alkali solution (10 units), and it also had added to it 
0.7 of a 1-per-cent solution of atropine sulphate. It 
was now found that the atropine augmented the action 
of the suprarenal extract five-fold, in the same way as it 
augmented the action of the azur dye — that is to say, 
with suprarenal extract by itself, and no atropine, the 
10 cc. of jelly, if it contains alkali to the extent of 
10 units, must contain at least 0.05 gramme of dried 
suprarenal extract before the earliest sign of cell- 
division can be induced in ten minutes. To obtain well- 
marked divisions the jelly should contain 0.2 gramme 
of the extract. 

If atropine is added, however, in the strength of 

1 Some 100-per-cent suprarenal extract has been purposely allowed to 
become infected, when it induced divisions in lymphocytes (figs. 103, 104). 
Control tubes of extract not so infected had not this action. 



310 THE CAUSE OF HEALING 

0.007 gramme of atropine sulphate to the 10 cc. of 
jelly which has 10 units of alkali, divisions in lympho- 
cytes can be induced if the jelly also contains no more 
than 0.01 gramme of dried suprarenal extract. Once 
more we tried to induce divisions with the alkaloid by 
itself, but failed; and yet it augmented the action of 
the extract five-fold. In addition to this augmentation 
it induced asymmetrical mitoses (fig. 105). 

To recapitulate: Extract of suprarenal gland of 
certain strength will induce by itself mitotic divisions 
in lymphocytes; and if more of it is made to diffuse 
into cells, it will also cause leucocytes to divide. If a 
lower concentration is tried, however, it will not induce 
divisions in the experimental ten minutes unless (1) it 
has become putrid, (2) its action is augmented by 
atropine. In both the latter circumstances asym- 
metrical mitosis may be seen. 

Other extracts of dead tissues were then tried; but 
they would not, by themselves, induce divisions in the 
experimental ten minutes. Realising that this might 
be due to the detrimental experimental conditions 
(corollary 2), we tried them again with atropine to 
augment their action. Now, as surmised, all the 
extracts of dead tissues which we tried induced 
divisions in lymphocytes on the microscope slide. To 
induce divisions in polymorphonuclear leucocytes with 
them is much more difficult, as atropine does not appear 
to augment their action so much with these cells. 

The following table gives the strengths of the 
various extracts which, with 1 cc. of alkali (10 units) 
and . 007 gramme of atropine sulphate, will induce 



AUXETICS ISOLATED 



311 




Fig. 102. — A dividing polymorphonuclear leucocyte induced by suprarenal 
extract alone. No stain. 





A 



Fig. 103. — Mitosis induced in a lymphocyte by suprarenal extract which 
had purposely been allowed to become putrid. No stain. 



r 



AUXETICS ISOLATED 



313 





Fig. 104. — Mitosis induced in a lymphocyte by suprarenal extract which 
had purposely been allowed to become putrid. No stain. 



Fig. 105. — Asymmetrical mitosis induced by suprarenal extract augmented 
by atropine. No stain. 



AUXETICS ISOLATED 315 

divisions in lymphocytes in the experimental ten 
minutes. 

Amount to be contained in the 10 cc. of jelly ^ 

Dried extract of Testis . . . 0.025 gramme 

" Pancreas . . . 0.025 

" Muscle . . . 0.025 

" Spleen . . . 0.01 

" Liver . . . 0.002 

Experimentally all these extracts were employed in a 
5-per-cent solution. Divisions in lymphocytes were in- 
duced with the first three by adding . 5 cc. of the solution 
to the 5 cc. of coefficient jelly, together with 0.7 cc. of 
1-per-cent solution of atropine sulphate, 1 cc. of the 
5-per-cent solution of sodium bicarbonate, and made up 
to a total of 10 cc. with 2.8 cc. of water. The jellies 
were boiled and films made from them in the usual way. 
It is obvious, therefore, that all the extracts contain 
some substance or substances which cause cell-division 
in lymphocytes and in leucocytes. To induce these 
divisions on the microscope slide in the experimental ten 
minutes, it is necessary to augment the action of the ex- 
tracts with atropine. Suprarenal extract, however, evi- 
dently containing more of the active substance than the 
others, will induce divisions without any augmenting sub- 
stance. Putrefaction will augment the power of the ex- 
tracts like the alkaloid, and it was presumed that this 
putrefaction had this effect through the presence of the 
alkaloids of putrefaction. This point, however, was not 
investigated till afterwards, as we were immediately 
concerned in finding out if possible what the agents 
were in these extracts of dead tissues which cause the 
division of white blood-corpuscles. 



316 THE CAUSE OF HEALING 

There were two ways in which we might attempt to 
isolate this active principle from the extracts. We 
might analyse them and try the different substances one 
by one. These analyses had, however, often been done 
before, and it was considered better, in the first instance, 
to try the well-known constituents of these extracts to 
see if they would induce cell-division before we under- 
took to analyse the extracts ourselves. 

We need not detail the vicissitudes of this research, 
which occupied a long time. The constituents of the 
extracts of the body are well known. It may be 
remembered that the active principle in the extracts is 
evidently thermostable, and remains in solution after 
most of the proteins have been precipitated by heat. 
We tried certain salts, and other substances, and we 
have also tried urea, and at last hreatin (C 4 H 9 N 3 2 ) 
was found to be a substance which will induce divisions 
in lymphocytes (fig. 106) and leucocytes (fig. 107). 
Kreatinin (C 4 H 7 N s O) is not effective in the experi- 
mental ten minutes; but xanthin (C 5 H 4 N 4 2 ) is if 
its action is augmented by atropine. 

The following table gives the strengths of kreatin 
and xanthin required to be contained in the 10 cc. of 
jelly in order to induce divisions in lymphocytes in the 
experimental ten minutes, no atropine being employed, 
but the jellies contained 1 cc. (10 units) of alkali 
solution. 

Kreatin. 

0.02 gramme . . . No mitosis seen. 

0.04 ... Early mitosis. 

0.75 . . . Well-advanced divisions. 



KREATIN AND XANTHIN 



317 




Fig. 106. — Mitosis induced in a lymphocyte by kreatin. No stain or extract. 






Fig. 107. — Division in a leucocyte induced by kreatin. No stain or extract 



KREATIN AND XANTHIN 319 

With atropine : 

Kreatin . 

0.005 gramme . . . No mitosis. 

0.01 " ... Mitosis. 

0.02 . . . Well-advanced divisions. 

Employing xanthin the presence of atropine is necessary : 

Xanthin. 

0.002 gramme . . . Early mitosis. 

If the jelly contained saturated solution 1 of xanthin, 
well-marked figures were seen. 

We had now succeeded in inducing the reproduction 
of leucocytes and lymphocytes — first by the aniline dye 
azur, then by a substance contained in the extract 
of suprarenal gland, and by our experiments we were 
able to infer that this substance is contained in the 
extracts of other dead tissues. It has just been shown 
that this inference is correct, because cell-division can 
be induced by the crystalline extractive kreatin, which 
is a constituent of the remains of all dead tissues. So 
far, of course, we had only induced divisions with these 
substances in vitro; but, as already pointed out, the 
cells are under very detrimental conditions while being 
experimented with, and it is more than probable that, if 
they will divide in response to these substances in vitro, 
they will more readily respond to them in vivo. 

Healing is caused by the proliferation of leucocytes and 
lymphocytes, and, judging from the in-vitro experimenta- 
tion, this proliferation is evidently induced by kreatin and 

1 Xanthin is sparingly soluble. 



320 THE CAUSE OF HEALING 

xanthin. Hitherto it has been generally supposed that 
the cell-proliferation of healing is due to some inherent 
propensity on the part of the cells to divide; but now 
it is clear, from in-vitro experimentation, that these 
cells divide when they absorb a definite quantity of a 
chemical agent, and two of these auxetics are kreatin 
and xanthin, which are contained in the remains of 
dead tissues. When a tissue is damaged anywhere, 
cell-death is occasioned, and the dead cells liquefy. 
The products of this death have as constituents the 
extractives kreatin and xanthin, and we know that the 
neighbouring living cells must absorb the liquefied 
remains of their dead neighbours, for it has been shown 
that the diffusion of substances into living cells is a 
physical process over which the cells themselves can 
exercise no control. When a tissue is damaged, there- 
fore, the direct result of that damage will be to make 
the neighbouring living cells reproduce themselves in 
response to kreatin and xanthin, and bring about the 
cell-proliferation of healing. 

Here, then, is the solution of the first part of our 
problem. We now know the nature of the physiological 
cause of the cell-proliferation of healing, and we submit 
that this knowledge reveals a fresh vista in pathology. 

But it must not be supposed that kreatin and 
xanthin are the only agents contained in the remains 
of dead tissues which cause cell-reproduction. They 
are two of the active principles which we have so far 
succeeded in isolating. It is probable that there are 
others; in fact, we know that there must be. Supra- 
renal extract will induce divisions very readily, and the 



THE NH, GROUP 321 



amount required to do so is so small that the kreatin 
and xanthin which it contains will not account for the 
divisions it induces. These bodies are amido-acids, and 
we think that the NH 2 group of the molecules may be 
responsible for the auxetic action. In this respect it 
is interesting to note that alkaloids, which augment the 
action of auxetics, are compound ammonias; but it 
must be remembered that we have never yet been able 
to induce a division with an alkaloid by itself, although 
we have tried literally hundreds of times. 

In the next chapter we shall show that there is 
another great and very important source of the " causes 
of the cell-proliferation of healing" contained in a 
substance we call "globin," a his tone derived from 
haemoglobin. 



CHAPTER XIV 

THE AUXETIC ACTION OF GLOBIN 

The fact that in-vitro experimentation has shown that 
cell-division is directly caused by certain constituents 
of the soluble remains of dead tissues made us consider 
the possibility that there might be other sources of 
these or similar agents. It was remembered how 
frequently old chronic ulcers, when they heal, leave 
the tissue pigmented, and it was considered possible 
that this pigmentation might in some way be asso- 
ciated with the healing process and its cell-prolifera- 
tion. The pigment in ulcers is supposed to be derived 
from haemoglobin. 

Melanotic sarcoma is generally accredited to be 
the most prolific of all malignant growths. It is 
characterised by the pigmented cells of which it is com- 
posed. We have not been able to obtain a case of 
melanotic sarcoma, for such cases are rather rare, but it 
is generally the case that the pigment is contained in 
the cytoplasm of the malignant cells. One of the 

322 



MALARIA PARASITE 323 

commonest sites of melanotic sarcoma is in the choroid 
coat of the eye, where the cells are normally pigmented. 
The pigment of these cells is called melanin, and it 
is supposed to be derived from hemoglobin. 

Professor Ronald Ross suggested that some experi- 
ments might be made with auxetics on the malaria 
parasite, and in one case a "crescent" was apparently 
made to flagellate prematurely with a jelly containing 
azur dye, extract, and atropine, although repetitions 
of the same experiment were not successful. Still, 
the consideration of the life-history of the malaria 
parasite has been — as it turns out — germane to our 
researches. The parasite enters the body from the 
mosquito as a minute unpigmented amoebula, which 
straightway enters a red blood-corpuscle. While in 
the red cell it gradually becomes pigmented, and it 
proliferates by exporulation. The daughter parasites 
have no pigment until they enter fresh red cells, 
when in their turn they become pigmented and ulti- 
mately proliferate again. 

There is the so-called sexual form of the cycle, 
however, which probably does not proliferate within 
the body. The crescent or gametocyte only pro- 
liferates after the blood containing it has been shed. 
The crescent is also deeply pigmented; and it is a 
most interesting point to remember that when the 
crescent stage of the parasite is reached, the red cell 
appears to be depleted of haemoglobin, and merely 
surrounds the parasite as an empty cell. The parasite, 
when it has reached the crescent stage, has apparently 



324 THE AUXETIC ACTION OF GLOBIN 

devoured all the haemoglobin; the haematin derived 
from the haemoglobin has collected in the parasite as 
a pigment known as melanin; and the parasite will 
no longer proliferate until the blood is shed. // the 
blood is shed, however, whether it is shed on to a 
microscope slide or into the stomach of the mosquito, 
the parasite again becomes prolific almost immedi- 
ately, and flagellation occurs. 

Now, when blood is shed, no matter how it is shed, 
whether it be on a microscope slide or into the 
stomach of the mosquito, haemoglobin must be set 
free, for the red corpuscle is a very delicate cell, and 
many of them must be ruptured when any injury 
occurs in a tissue. The question therefore arises, Does 
haemoglobin have any function in inducing the pro- 
liferation of the malaria parasite ? From circumstantial 
evidence it would appear that it does, for so long as 
the parasite is absorbing haemoglobin from the red 
cell in which it lives, so long will it continue to 
proliferate by exporulation ; but when it has finished 
the contents of the cell, proliferation ceases until 
more haemoglobin can be absorbed by it when the 
blood is shed. 

In the malaria parasite, in the cells of melanotic 
sarcoma, and in the neighbourhood of old healing 
ulcers the haemoglobin is evidently decomposed because 
the haematin collects as insoluble pigment. 

Haemoglobin is fairly soluble, but when it is de- 
composed into haematin and globin the haematin is 
insoluble in water except in the presence of dilute 
alkalies. Globin is readily soluble. Hence it cannot 



EXPERIMENTS WITH GLOBIN 325 

be the haematin part of the haemoglobin molecule which 
has any function in causing proliferation; it must be 
the globin part if it is either of them. 

In the first instance we tried the effect of haemo- 
globin on blood-cells. Jellies were made which con- 
tained 1 cc. (10 units) of alkali solution, and after they 
had been boiled various quantities of a saturated solution 
of crystalline haemoglobin were added before the jellies 
cooled too much for them to set on a slide. But haemo- 
globin never induced divisions in lymphocytes or 
leucocytes in the experimental ten minutes. Nor did 
it excite amoeboid movements in them. 

We next made a saturated solution of haemoglobin 
and then boiled it, thereby decomposing it and pre- 
cipitating the haematin. The filtrate is a straw-coloured 
liquid when it is dilute. It was evaporated down by 
prolonged boiling, and at the saturation point, which 
is about 4 per cent, the solution becomes a deep red 
colour. On evaporation to dryness, a sticky residue 
remained. Very little is known about globin. For 
years it was thought to be a globulin, but this has 
been shown not to be the case. Globin is a histone — 
a protein which is not precipitated by boiling. In the 
dry state it is a glutinous mass of a deep brick-red 
colour, and it has a characteristic sweet smell some- 
thing like licorice. If it is very dry, globin can be 
ground into a brown powder. It is at all times ex- 
tremely hygroscopic, and therefore if it is not kept in 
solution it must be placed either in a desiccator or in 
sealed tubes. If it is kept in solution and exposed 
to the air, it rapidly decomposes owing to putrefaction, 



326 THE AUXETIC ACTION OF GLOBIN 

and gives off a foul smell, reminding one of that of 
the alkaloid neurine. 

Jellies were made which contained various strengths 
of globin, and, of course, certain quantities of alkali 
solution were also added. It was found that globin 
by itself would never induce divisions in lymphocytes 
in the experimental ten minutes, so we tried it again 
with the addition to the jellies of 0.7 per cent of 
atropine sulphate, and then globin induced divisions 
in lymphocytes (figs. 108, 109). This is the best strength 
to employ: In 10 cc. of jelly containing 10 units of 
alkali and . 007 gramme of atropine there should also 
be 0.0025 gramme of globin. The best divisions are 
obtained with . 025 gramme of globin ; but if the 
content of it exceeds . 05 gramme, the cells appear 
to be poisoned, because they shrivel up and frequently 
burst. 

Some globin in solution (1 per cent) was allowed to 
putrefy for a fortnight, and, like extracts of dead tissues, 
it was then found that its action was so augmented that 
it also would (in the strength of 0.005 — or better 0.01 
gramme — in the 10 cc. of jelly) induce divisions by 
itself (without atropine) in the experimental ten minutes 
(fig. 110). 

When putrefaction occurs in a solution of globin 
a precipitate falls, and yet it is now more effective in 
inducing divisions than it was before. It is clear, 
therefore, that it is not actually globin which induces 
divisions, but it is some constituent of it which is effec- 
tive. Putrefaction decomposes globin, and the active 
agent plus some augmenting substances are produced. 



EXPERIMENTS WITH GLOBIN 



327 






Fig. 108. — Mitosis in a lymphocyte induced by globin augmented by 
atropine. No stain, extract, or kreatin. 




Fig. 109. — Asymmetrical mitosis induced by globin augmented by atropine. 
No stain, extract, or kreatin. 



EXPERIMENTS WITH GLOBIN 329 




Fig. 110. — Mitosis induced in a lymphocyte by means of decomposed globin 
solution. No stain, extract, kreatin, or atropine. 



EXPERIMENTS WITH GLOBIX 331 

It must be understood that if the jelly on which the 
cells are resting contains . 02 gramme, or more, of 
o*lobin, the red cells become distorted and the white 
cells are killed without divisions being induced in 
them. 

We think that it should be mentioned that it is 
quite within the realms of possibility that the malaria 
parasite proliferated in response to the active agent 
contained in globin; but although we have tried a few 
experiments to endeavour to prove the point, we have 
not succeeded in determining it. Malarial crescents 
frequently flagellate in any case within ten minutes of 
their being shed; and although we have mixed the shed 
blood containing them with citrated solutions of globin, 
it has been impossible for us to satisfy ourselves that 
the flagellation has been accelerated by its action. In 
the cases of malaria at our disposal there have not been 
a very large number of parasites in the blood, and time 
was lost during the experiments in finding them. 
Hence we cannot speak definitely on this point, but 
it was the consideration of the life-history of the malaria 
parasite which was the chief factor which led us to 
investigate the auxetic property of globin; and there is 
no doubt whatever that globin contains some auxetic, 
although it is not so powerful as that contained in 
suprarenal extract. 

Globin contains no kreatin so far as we can ascer- 
tain, and the solution of globin which we have used is 
free of hsematin, as proved by spectroscope examina- 
tion, and there are only traces of lipochrome. What 
the exact nature of the auxetic substance contained in 



332 THE AUXETIC ACTION OF GLOBIN 

globin is we do not know, but possibly it is allied in 
some way to the molecules of kreatin and xanthin. It 
should also be remembered that we do not know what 
the substance is in the azur dye which induces divisions. 
We think that they will not be difficult to isolate; but 
we ourselves do not feel competent to undertake 
chemical analyses of this nature. 



CHAPTER XV 

THE PROOF THAT THE REMAINS OF DEAD TISSUES AND 
GLOBIN CONTAIN THE CAUSES OF THE CELL- 
PROLIFERATION OF HEALING AND OTHER CELL- 
REPRODUCTION EXPERIMENTATION in vivo CON- 
FIRMS in-vitro observations — the cause of 

BENIGN TUMOURS 

The foregoing experiments show that some of the 
causes of human cell-division are now known. On 
the stage of the microscope white corpuscles can be 
made to undergo the stages of cell-division in direct 
response to certain chemical agents, two of which have 
been isolated, and which can be employed in crystalline 
form to induce cell-division. What is far more im- 
portant, however, is the source whence these chemi- 
cal substances are derived. They are contained in the 
soluble remains of dead tissues. Another source of the 
cause of cell-division is in globin, which is derived 
from the decomposition of haemoglobin. 

It should be remembered that so far the experimen- 
tation has been confined to testing the action of the 
active substances and the sources of them on individual 

333 



334 THE CHEMISTRY OF PROLIFERATION 

cells which have been removed for the purpose from 
the body; and, as already pointed out, the cells in this 
in-vitro experimentation are not by any means in 
conditions similar to the natural ones under which they 
normally exist. Still there is no question whatever 
that the cells do divide in response to these agents ; and 
if they will do so under detrimental experimental 
conditions, it is obvious that they will be far more 
likely to divide and respond to the same agents ; in their 
normal conditions. The agents we know do not exist 
in the body, and therefore it is practically a certainty 
that these substances will cause proliferation there. 
For reasons already given, on the microscope slide one 
cannot induce more than one generation of cells by 
chemical agents, because premature death cannot be 
prevented; but in the body the premature death need 
not necessarily occur, for its cause is absent, and hence, 
provided the causes of cell-division are being constantly 
supplied to cells, generation after generation must be 
produced. 

On the microscope slide cells will not divide, so far 
as can be seen, unless they absorb definite quantities 
of the agents which cause cell-division. We do not 
say that there are no other substances which cause cell- 
division besides those which have been mentioned — 
in fact, we know that there must be others; but what 
we think is now becoming evident is the fact that cells 
will not divide at all unless they receive some chemical 
agent which makes them do so. That is to say, we 
think that there is strong evidence in support of the 
view that cell-division in the body is entirely caused by 



AUXETICS ARE NECESSARY 335 

chemical agents ; and if these agents are not present, 
there will be no cell-division. 

In the case of leucocytes. For nearly a century 
and a half these cells have been observed in the blood. 
Every doctor and student of medicine must have seen 
them alive repeatedly, and yet not a single person had 
ever seen them divide. Now, however, if one makes 
them absorb certain chemical agents the cells divide im- 
mediately; and what is more, we have shown that the 
rapidity of onset and the time occupied by each division 
varies directly with the quantity of the substances 
absorbed by the cells. Cell-division appears to be a 
physical phenomenon which can be measured in the case 
of each cell in proportions of grammes of the chemical 
auxetics absorbed by them. We have shown how it 
can be set down as a simple mathematical equation. 
It must be admitted that in spite of the fact that 
blood-cells have not been seen to divide without an 
auxetic, there is no actual proof that a cell cannot 
divide without one. It has yet to be proved that 
human leucocytes have no inherent power to multiply 
"when they feel so inclined," but it is a remarkable 
thing that no single leucocyte, out of the many millions 
which have been seen by men, should ever have de- 
veloped this inclination during nearly a century and a 
half. On the other hand, we know that if we cut our 
fingers and so produce the remains of dead tissues 
containing kreatin and xanthin, proliferation of leuco- 
cytes occurs immediately; and the greater the injury, 
the greater the cell-proliferation. 

We think that if the problem is carefully con- 



336 THE CHEMISTRY OF PROLIFERATION 

sidered, and, better still, if these mitotic divisions 
are actually seen as they occur in response to chemical 
agents, it will be appreciated that there is a strong 
probability that cells only divide when they are made 
to do so by an exciter of reproduction. 

The active auxetics are contained in "the remains 
of dead tissues." Globin is in reality "the remains of a 
dead tissue," for it is obtained by the decomposition 
of haemoglobin, and haemoglobin is contained normally 
in living red cells. Doubtless the constituents of the 
molecules of kreatin, xanthin, and the active principle 
of globin are present in living protoplasm; but they 
may not be present, presumably, in the same combina- 
tion or form as they exist in kreatin and xanthin. 
Possibly it is only after death that these substances 
are produced, in which case it would follow that a cell 
will not reproduce itself by virtue of the constituents 
of its own living protoplasm; but it is necessary for it 
to absorb fresh active agents from the dead remains of 
its neighbours. 

Many points are now explained. When it is re- 
quired that an indolent healing surface shall heal well, 
we scarify it, as exemplified in the operation of Thiersch 
grafting. If a fractured bone will not unite, the ends 
are rubbed together or actually "freshened" by opera- 
tion, to produce callus; and callus is really a tissue 
made by the proliferation of cells. When we scarify 
or freshen a surface, we merely cause destruction, and 
thereby set free exciters of reproduction. If a part of 
the body is bruised, haemorrhage occurs; and, as is 
shown by the pigmentation, the haemoglobin set free 



CELL-DEATH INDUCES CELL-BIRTH 337 

from destruction of red cells which have been shed 
into the injured tissues is decomposed, and globin is 
thus locally produced. The cell-proliferation of healing 
must then occur in response to it, and the remains of 
other tissues which have been killed in the injury. 

The proliferation of cells, however, is not confined 
to the cell-proliferation of healing. It will be shown 
that epithelial cells will also respond to auxetics, and 
probably some if not all other cells also respond to 
the soluble remains of their neighbours by reproducing 
themselves. It is true that globin does not exist in 
the cornea, for here there is no blood supply, and con- 
sequently no haemoglobin until some time after the 
injury. Still, if the cornea is injured the corneal cells 
must be injured, and the cell-proliferation of healing 
occurs in response to the remains of the injured cells. 

Irritation is always followed by cell-proliferation. 
Irritation means damage, and damage means cell-death. 
Cell-death sets free kreatin, xanthin, and other auxetics, 
and the cell-proliferation is caused by their absorption 
by the neighbouring living cells. The greater the 
damage, the greater will the cell-proliferation be. 

Cell-division is apparently an automatic phenom- 
enon — not in the sense that it is due to some in- 
trinsic function or duty of a cell's protoplasm, but 
automatic in that the death of one cell will cause the 
reproduction of its living neighbours. If we may 
speak of the act of cell-division by mitosis as the 
"birth" of cells, then we may say that the number 
of births of cells in the body depends on the number of 
deaths. The greater the number of deaths, the greater 



338 THE CHEMISTRY OF PROLIFERATION 

•the number of births. If an individual cell dies, its 
death causes its neighbours to multiply to supply the 
deficiency; but if the cell-death is extensive owing to 
damage, the proliferation of those cells which have not 
been killed will also be extensive, and this proliferation 
will now be extended to that of the white blood-cor- 
puscles which have been shed during and after the 
injury; and the result will be the cell-proliferation of 
healing. 

Judging from the experiments which have been 
made, it may also be assumed that since the number 
of cell-births depends upon the number of cell-deaths, 
and since an increase in the number of births must 
increase the number of deaths, it follows that the 
number of deaths must also depend to some extent 
on the number of births. Presumably, if once cell- 
division is set going in a tissue or in a part of a tissue, 
that cell-division will go on increasing until something 
restrains it. Elimination from a tissue of tissue fluids 
would restrain it; for if the soluble remains of dead 
tissues become quickly eliminated, the diffusion of the 
constituents of these fluids into the cells would also be 
arrested, for that diffusion varies directly with the 
factor time. In a damaged tissue the vessels and 
lymphatics are also damaged, and elimination may be 
impaired ; hence the remarkable cell-proliferation which 
leads to "granulation tissue." In an injury of any part 
except the cornea, coagulation of the shed blood occurs ; 
the red cells become laked, and ultimately the haemo- 
globin is evidently decomposed, as evinced by the 
pigmentation which will always be seen even in a 



ORIGIX OF BEXIGX GROWTHS 339 

bruise. The globin so produced will assist in pro- 
moting the cell-proliferation of healing. 

Such is the explanation of the cause of cell-division 
in the human body as demonstrated by in-vitro experi- 
mentation. But we think that we may go farther, 
and suggest that the initial multiplication of the cells 
in the human embryo may also be caused by a chemical 
auxetic. Spermatazoa contain extractives. Possibly 
it is these extractives, set free from this spermatozoa, 
which, after fertilisation, give rise to the subsequent 
cell-division in the ovum from which the embryo is built 
up. Once the cell-division has started, it will go on in 
response to the cell-deaths which sooner or later must 
occur. 

As we have pointed out, kreatin is not by any 
means the only auxetic contained in the remains of 
dead tissues, and it is yet to be proved that there is 
not some specificity in cell-reproduction due to some 
at present unknown substance. We know from the 
study of heredity that certain characteristics are car- 
ried in the ovum and in the spermatozoon, and if they 
are so carried, doubtless other chemical auxetics, far 
more complex than kreatin, may be carried too. 

In the meantime we think that the knowledge that 
dead tissues cause cell-proliferation is sufficient to give 
an inkling as to the cause of benign growths. A 
sudden cell-death occurring in a tissue will cause pro- 
liferation of neighbouring cells. Of course, if the 
initial cell-death is extensive, the cell-proliferation of 
healing will occur which ultimately leads to the pro- 
duction of connective tissue, which in itself may 



340 THE CHEMISTRY OF PROLIFERATION 

prevent undue extension of the proliferation of the 
normal tissue-cells. But supposing for some reason, 
such as a slight injury, a local cell-death takes place: 
it would cause increased proliferation of local cells, 
and so form the basis of a tumour. Once this growth 
is started, it will go on until, by causing "irritation" 
or, to be more accurate, extensive cell-death, it may 
now induce the cell-proliferation of healing round it, 
and so, by the formation of connective tissue, cause its 
progress to be arrested by a capsule. A benign tumour 
is probably due merely to some localised cell-death in 
the first place, and it is remarkable how frequently 
there is a history of injury in these cases. But there 
is also no doubt that the onset of benign growths, and 
other cell-proliferation too for that matter, must be con- 
trolled to some extent by nervous influence. Possibly 
this nervous influence may be actuated by the nervous 
control over local elimination. Quite recently a paper 
appeared in The Lancet on a case of bilateral benign 
tumours; 1 and this can only be due to some central 
control over the local causes of cell-division. 

Fibroids of the uterus occur only during the years 
of menstrual activity. During this time the uterus 
periodically becomes enlarged, followed by reduction in 
size. This reduction and quiescence must be accom- 
panied by death of living cells, and presumably it is 
this death which, if elimination of the products of 
katabolism is impaired, may lead to excessive pro- 



1 See a paper on Bilateral Tumours by W. Roger Williams in The Lancet, 
Feb. 12, 1910. 



ULCERS TREATED WITH AUXETICS 341 

liferation of the remaining living cells, and so cause 
the growths known as fibroids. 

The foregoing conclusions and deductions have 
been arrived at from experimentation in vitro with 
individual cells. As pointed out in a former chapter, 
conclusions derived from in-vitro experimentation are 
not in themselves sufficient to prove a point. Because 
we can induce cell-division in individual cells on the 
microscope stage with certain chemical agents does 
not prove that the same division will necessarily occur 
in vivo in the same cells in response to the same 
agents. But, fortunately, in-vivo experimentation with 
these agents has not been impossible, and the proof 
that these agents, or rather some of them, do actually 
cause proliferation in the body is now at our dis- 
posal. In the wards of the Royal Southern Hospital 
at Liverpool cases of chronic callous ulcers of the 
legs were admitted, and have been treated in the first 
instance with saturated solutions of globin. The 
globin was applied to portions of the ulcers by dip- 
ping pieces of sterile gauze in the solution and applying 
it direct to the ulcerated surfaces. Granulations im- 
mediately appeared in response. In the short space 
of three or four hours a difference appeared between 
the extent of the granulations in the treated as com- 
pared with the untreated portions of the sores. In 
twenty hours the difference was marked. Granulomata 
have been produced in a day or two by means of 
globin. 

Others suggested that the proliferation was not 
necessarily due to the globin, but to the " irritation" 



342 THE CHEMISTRY OF PROLIFERATION 

of the gauze, in spite of the fact that ulcers have been 
treated with gauze all over, but only a part of them 
with globin added, and the proliferation occurred to 
the marked extent only where the globin was. We 
therefore discarded gauze or dressings altogether, and 
repeated the experiments. In a case where there were 
several ulcers on one leg the surfaces of them all were 
scarified, and small pieces of dried globin were " dotted" 
all over one ulcer. The cell-proliferation occurred to a 
marked extent in that ulcer, but only to a much less 
extent in the others which were not so treated. 

Globin thus applied to a healing surface causes a 
scab to form very rapidly (figs. Ill, 112), and the 
cell-proliferation goes on beneath it. This scab forms 
in an hour or two, whereas, if no globin is applied, it 
takes several days for a scab to form on an ulcer 
which has no dressing on it. Globin also causes ex- 
tensive proliferation of the epithelium from the sides 
of the ulcer. 

Unfortunately suppuration occurs under the scab, 
no matter how "clean" the ulcer may be when the 
globin is applied. The onset of suppuration, how- 
ever, has been delayed by preparing the globin with 
aseptic precautions throughout, thus: A solution of 
haemoglobin is decomposed by boiling, and filtered, and 
the globin solution is concentrated until it precipitates 
by further boiling. It is evaporated to dryness at a 
temperature of 60° C. and immediately sealed into sterile 
glass tubes. Even with these precautions, suppuration 
usually occurs under the scab in the course of a few 
days. The scab is then removed with fomentations, 



CLINICAL OBSERVATIONS 



343 




Fig. 111. — To show the way in which globin is ''dotted" over the surface of an ulcer. 




Fig. 112. — To show the scab formed by the application of globin to an ulcer. 



CLINICAL OBSERVATIONS 345 

and when the sore is clean it is once more scarified, 
and fresh sterile globin is again "dotted" over its 
surface. This procedure can be repeated until the 
ulcer heals. During the scarification it is better to 
draw blood. Latterly this treatment of ulcers has 
been improved by using powdered globin (five parts), 
mixed with two parts (by weight) of kreatin, a mix- 
ture which produces more marked proliferation than 
pure globin. 

Many ulcers have now been treated by this method, 
and we think that we can say safely that it causes 
more rapid healing of them than if they were treated 
in the usual way. Callous ulcers will usually heal 
by themselves if the limbs are kept at rest, and it 
was suggested to us that the cell-proliferation pro- 
duced by globin was in reality due to the fact that the 
patients were kept in bed. This suggestion was dis- 
proved, however, by the production of extensive pro- 
liferation in one part of an ulcer by means of globin 
in a patient who was made to walk about during the 
treatment. Lastly, granulations have been induced by 
extracts of suprarenal gland. 

It should be mentioned that globin, kreatin, etc., 
when applied to a healing surface will not only cause 
proliferation during the application; but once the mul- 
tiplication has started, it will continue "automatically," 
even though the application of the auxetic is discon- 
tinued. This point has frequently been seen during 
the experimentation with ulcerated legs, and it is proof 
that the proliferation of cells is "automatic." There 
can be no doubt that once proliferation is started 



346 THE CHEMISTRY OF PROLIFERATION 

in an ulcer, an increased number of deaths is occasioned, 
which in its turn still further increases the proliferation, 
as seen in the ulcers once treated with globin. 

The application of dry globin to a scarified sore has 
elicited the interesting fact that it will convert the dark 
venous blood drawn by the scarification into the bright 
and red arterial variety, and the scars resulting from 
the treatment appear to be exceptionally firm and un- 
likely to break down again. 

This form of treatment, however, must be carried 
out with care, and suppuration not allowed to continue 
for long in the presence of an auxetic, for, as will be 
shown in the next chapter, there is a possibility of 
malignant proliferation occurring in place of the normal 
one if the products of decomposition become pent up 
in the neighbourhood of proliferating epithelial cells. 

These experiments afford conclusive proof that the 
cell-proliferation of healing can be caused by the chem- 
ical auxetics, kreatin and globin, and that the deduc- 
tions made from the prolonged experimentations with 
the in-vitro method described in this book are correct. 
The possibility of the mitotic divisions induced on the 
microscope slide being in the nature of "freaks" or 
being due to death-struggles is disproved. As a matter 
of fact, these possibilities practically fell to the ground 
when mitoses were induced by extracts of dead tissues. 
One could conceive that a purely artificial substance 
like azur dye might cause mitosis by exciting the cells 
greatly just before death; but we think that in all 
probability the aniline dye contains some constituent 



CLINICAL OBSERVATIONS 347 

which possibly resembles the molecules of the natural 
auxetics. 1 

The fact that the cell-proliferation of healing is 
caused by chemical agents contained in the soluble 
remains of dead tissues will, we confidently believe, 
be the means of solution of many problems which at 
present confront the investigator in pathology and 
perhaps in physiology also. It is a fact about which 
there can be no doubt whatever. 

1 The formula of azur dye {Cent. f. Bakteriologie, Bd. xxix., 1901) is: 

S0 2 
(CH 3 ) L N\ /\ /\ - /\ ^N(CH 3 ) 2 .C1 




CHAPTER XVI 

THE AUGMENTED DIVISIONS INDUCED BY PUTREFACTION 
OF THE EXTRACTS ARE DUE TO THE ALKALOIDS OF 

PUTREFACTION A THEORY THAT CARCINOMA AND 

LYMPHADENOMA MAY BE CAUSED BY THE MIX- 
TURE OF THE AUXETICS OF CELL-PROLIFERATION 
WITH CHOLINE OR CADAVERINE AN EXPLANA- 
TION OF THE AGE-INCIDENCE, METASTASES, AND 

OTHER FACTS KNOWN CONCERNING CANCER THE 

NECESSITY FOR A CRUCIAL EXPERIMENT TO PROVE 
THE THEORY 

In Chapter IX. it was pointed out that there is an 
intimate association between "chronic irritation" and 
the onset of cancer. As just shown, "irritation" means 
cell-death, and cell-death is followed by cell-prolifera- 
tion. When a tissue is the seat of chronic irritation, 
the cell-proliferation of healing must be going on in the 
damaged site owing to the presence of the remains of 
the dead cells. The proliferation occasioned by irrita- 
tion is in realitv due to the auxetics, some of which are 
kreatin, xanthin, and that contained in globin, which 
are set free by the death of some of the cells. This 
will explain why an ill-fitting boot will give rise to 

348 



THE AUXETICS OF IRRITATION 349 

a "corn," and to the "induration" of a tissue which is 
under pressure or being chronically irritated. In reality 
"irritation" must be followed by chronic cell-prolifera- 
tion due to the auxetics produced. 

Now, the chief characteristic of cancer is that it 
consists of a growth of cells which are proliferating 
excessively. Every cancer is a growth which infiltrates 
the surrounding tissues; and this growth occurs pro- 
bably in every instance in a site in which there is 
chronic irritation — or rather where there is chronic 
cell-proliferation of healing due to auxetics. 

One may suggest, therefore, that since the prolifera- 
tion of chronic irritation is due to the auxetics produced 
by cell-death, the proliferation of cancer is also associated 
with them. The proliferation of chronic irritation, 
however, is a normal one, whereas that of cancer is 
a malignant one. If the cause of the normal prolifera- 
tion is removed, then ultimately proliferation ceases; 
but if the irritation which predisposed to cancer is 
removed, the malignant cells appear to continue to 
multiply until the patient dies. Yet cancer-cells are 
cells of the body. They are not foreign parasites, and 
hence it may be that in a cancerous growth there is 
some other factor in addition to the normal ones. 
Therefore it may also be suggested that the onset of 
cancer in a normal healing site may be brought about 
by the presence of another agent in addition to the 
normal auxetics produced by cell-death. 

Now let us return to the "augmenting" of the 
action of auxetics in promoting cell-division by putre- 
faction and by the alkaloid atropine. It is well known 



350 THE PROLIFERATION OF CANCER 

that certain putrefactive bacteria in decomposing dead 
organic structures produce ptomaines and leucomaines. 
These substances are in the nature of alkaloids. The 
following are common ones: 

Choline . . . . C 5 H 15 NO r 

Cadaverine . . . C 5 H 14 N 2 . 

Neurine .... C 5 H 13 NO. 

Putrescine . . . C 4 H 12 N. 

Choline will, like other alkaloids, excite amoeboid 
movements in leucocytes and lymphocytes, and so will 
cadaverine. In fact choline is just as effective as 
atropine in this respect. The best strength of choline 
to employ to excite amoeboid movements in leucocytes 
and lymphocytes is one in which 10 cc. of jelly con- 
tains 0.01 gramme of the alkaloid in addition to the 
10 units of alkali. Choline, however, is not very poison- 
ous to leucocytes, and even . 04 gramme will not kill 
them. Cadaverine also excites leucocytes, and 10 cc. 
of a jelly containing 1 cc. of a 1-per-cent solution of 
it is suitable for this purpose if 10 units of alkali are 
also present, the jelly-film being examined, of course, 
at the room temperature. 

It may be remembered that it was through the 
accidental putrefaction of the extract of suprarenal 
gland that we were enabled to induce divisions with 
it by itself for the first time, and we now know that 
the reason for this was that the putrefaction produced 
the alkaloids choline and cadaverine in the solution 
of the extract, and that they, like atropine, greatly 
augment the action of auxetics in inducing cell-division. 



THE EFFECTS OF ANIMAL ALKALOIDS 351 

In order to prove this point we now, in the first instance, 
used these pure alkaloids, choline and cadaverine, 
added to the extracts, and afterwards we combined 
them with kreatin and xanthin to induce augmented 
divisions. 

If a jelly contains 0.01 gramme of choline and 
10 units of alkali solution, divisions in lymphocytes 
can be induced if only . 02 or even 0.01 gramme of 
kreatin is present (fig. 113). In fact, this alkaloid of 
putrefaction choline, like atropine, augments the action 
of auxetics about five-fold. 

Using cadaverine in the strength given above, 
divisions in lymphocytes were induced if the jelly con- 
tained only 2 cc. of a 1-per-cent solution of kreatin. 

It has already been mentioned that a mixture of 
atropine and an auxetic will give rise to asymmetrical 
mitosis in lymphocytes, and we have also found that 
these remarkable mitoses also are frequently induced 
by the augmenting action of choline and cadaverine 
(figs. 114-16). This point is of great importance, 
because it is well known that asymmetrical mitoses 
are frequently seen in cancerous growths. 

So far the augmented divisions had only been 
induced in lymphocytes. It is true that there is a form 
of cancer which occurs in the lymphocyte class of 
cells of the lymphatic glands (lymphadenoma) ; and if 
it is a criterion that because a lymphocyte divides by 
an augmented asymmetrical division it is necessarily 
malignant, 1 then the combination in certain proportion 
between the causes of the proliferation of healing plus 
an alkaloid of putrefaction like choline must be a cause 

1 It has not been proved to be a criterion. 



352 THE PROLIFERATION OF CANCER 

of lymphadenoma. In connection with this it is 
interesting to note that many years ago Trousseau 1 
stated in his book that lymphadenoma often follows 
on a suppuration focus, and this view is upheld by 
many to this day. At the same time it must be re- 
membered that no alkaloid has yet been made to induce 
a division by itself; it is essential for an auxetic to be 
present also. Alkaloids appear to be augmenters only 
of cell-division. 

But our object was, if possible, to find the cause 
of carcinoma, and we therefore tried to see if our 
chemical agents would induce divisions in epithelial 
cells. Considerable difficulty was met in investigating 
this point. Epithelial cells will not live long in vitro; in 
fact, they usually die in a few moments, as far as can be 
seen. But at last we did succeed in inducing an early 
mitotic figure in two epithelial cells (as shown in the 
photographs, figs. 117, 118) from the vaginal secretion. 
We did not succeed in inducing the divisions with an 
entirely "natural" agent, for epithelial cells evidently 
require more auxetics than even leucocytes. The 
figure induced was seen when the epithelial cells were 
placed on a powerful jelly which contained azur stain, 
putrid extract of suprarenal gland, and atropine. 
In vivo, also, epithelial cells undoubtedly proliferate 
in response to globin and kreatin. 

The fact was, therefore, proved that epithelial cells 
respond to the chemical exciters of reproduction, and 
it is possible that they may be subject to the same 
conditions as lymphocytes, and only respond to them. 

1 Trousseau's Clinical Medicine (Sydenham Society), 1872, vol. 5, p. 207. 



AUGMENTORS OF AUXETICS 353 





Fig. 113. — Mitosis induced by a mixture of kreatin and choline. No stain, 
extract, or atropine. 





Fig. 114. — Asymmetrical mitosis induced in a lymphocyte by a mixture 
of suprarenal extract and globin, augmented by choline. No stain or 
atropine. 

23 



AUGMEXTORS OF AUXETICS 355 










Fig. 115. — Mitosis in a lympnocyte induced by globin and choline. No 
stain or other auxetic. 




Fig. 116. — Mitosis induced in a lymphocyte by suprarenal extract and chol- 
ine. No stain or other auxetic. 



AUGMENTORS OF AUXETICS 357 




Fig. 117. — Mitosis induced in an epithelial cell by a mixture of stain and 

extract. 





Fig. 118. — Early mitosis in an epithelial cell from the vagina induced by 
. stain and extract. 



AUGMENTORS OF AUXETICS 359 

The action of these auxetics upon lymphocytes 
is greatly augmented by the alkaloids choline and 
cadaverine, which are produced in a solution of an 
extract of a dead tissue as it decomposes. We now 
made some experiments to see if putrefaction also 
augmented the auxetic action of globin, and a solution 
of it was therefore allowed to decompose at the room 
temperature for about three weeks. A solution of 
globin of no matter what strength will not (as already 
noted) induce divisions by itself in ten minutes; it is 
necessary to add atropine. But when a 1-per-cent 
solution of globin had decomposed, it was found that 
now it would induce divisions in lymphocytes by itself 
in the experimental ten minutes. Whether this aug- 
mentation of the action of globin by decomposition 
is due to the production of choline and cadaverine or 
not, we are uncertain; for although we have tested 1 the 
decomposed solution for the presence of alkaloid, only 
a negative result has been obtained. This matter will 
require further investigation, for it is possible that other 
substances besides the alkaloids of putrefaction may 
augment the action of exciters of reproduction. We 
have so far obtained the best divisions by making up 
the 10 cc. of jelly with 0.5 cc. of a 2-per-cent solution 
of globin which had been kept open to the air of the 
room for three weeks. 

Decomposition of organic solutions which contain 
exciters of reproduction will augment the action of the 
latter agents up to as much as five-fold; and in this 
case the divisions induced in lymphocytes are fre- 

ir The iodine and the mercuric chloride tests were employed. 



360 THE PROLIFERATION OF CANCER 

quently of the asymmetrical variety. Cancer is a 
growth of cells which supervenes on an old irritated 
site where the cell-proliferation of healing has been 
going on for some time. Since cancer is an exu- 
berant growth of epithelial cells — which will respond 
to auxetics — it is obvious that the quantity of the action 
of the normal auxetics present must be augmented in 
some way so as to give rise to the exuberant prolifera- 
tion of malignancy. And lastly, the mitoses in a 
cancerous growth are frequently of the asymmetrical 
type. The combination of a normal auxetic plus an 
alkaloid of putrefaction and decomposition will cause 
not only augmented divisions, but it is important to 
note that these divisions tend to be asymmetrical in 
character. 

Having arrived at this stage of our researches, these 
new facts were carefully considered to see how they 
harmonised with the well-known features which are 
associated with cancer, and we shall now discuss them. 

The fact that cell-division is caused by substances 
contained in the remains of dead tissue throws light 
on the age-incidence of the disease. 

In a paper published by us in The British Medical 
Journal on October 23, 1909, when we were aware of 
the fact that cell-division could be induced by an aniline 
dye, and that its action could be augmented (which 
was all we knew then) by the remains of a dead tissue, 
We appreciated that the remains of dead tissues might 
be a predisposing factor in the cause of carcinoma. We 
may as well quote the passage from that paper, for it 
shows the possible relationship between the remains of 



AGE-INCIDENCE 361 

dead tissues (the products of katabolism) and the age- 
incidence of cancer. 



The body is mainly composed of living cells, and 
they constitute an elaborate combination of living 
factors. We know that in certain tissues these cells 
are continually dying and being replaced, so that it 
is evident that birth and death must be going on 
incessantly in the body. What happens to the dead 
cells ? They of course liquefy and become dis- 
organised, and their constituents are presumably ex- 
creted or converted into other compounds. While 
this is happening it seems probable that some of the 
products of the remains of dead cells may be absorbed 
by their neighbours, for it must be remembered that 
the diffusion of substances into living cells appears to 
be a physical process over which they exercise no 
control. There are doubtless some cells which remain 
alive for long periods; for instance, it has been 
estimated (and we are informed that it is practically 
certain) that some cells of the central nervous system 
live throughout the life of a man. Many cells, how- 
ever, only live a very short time, the length of their 
lives perhaps varying in different parts of the body, so 
that the remains of dead cells are probably always 
present in the body fluids. In this connection, how- 
ever, we have to keep in mind the physiological curve 
expressive of the relationship between anabolism and 
katabolism. There are only three stages of life if it is 
viewed from this point of view, the first terminating at 
about the thirtieth year, when a man reaches his prime, 
and up to which period cellular birth must preponderate 
over its death-rate. For some years it may be sug- 
gested that anabolism and katabolism remain balanced ; 



362 THE PROLIFERATION OF CANCER 

and that after the age of 40, quite physiologically, so 
that nothing occurs to make a man aware of it 
physically, these conditions begin to be reversed and 
more of the products of katabolism — that is, the 
remains of the dead cells — tend to exist in the body- 
fluids than was the case before middle age. 

Here we have a fact incidental to the cancer period 
which suggests the possibility that these products of 
katabolism may in some way predispose to the onset of 
malignancy. It cannot possibly be suggested that they 
are the cause of the disease, for if such were the case 
everybody over the age of 40 would die of cancer; but 
assuming that some product of katabolism may possibly 
favor the onset of the disease, we may enlarge upon the 
speculation and say that it is a certain morphological (or 
chemical) element in a dead cell which may be the agent. 
For the sake of argument it may be derived from either 
the cytoplasm, the cell-wall, the nuclear wall, or the 
linin, or it may be the chromatin itself. 



It is now known, of course, that the products of 
katabolism actually contain causes of cell-reproduction ; 
and it follows that if it is correct that these products are 
in excess after the age of 40, there must be, ceteris 
paribus, a greater inclination to cell-proliferation in a 
tissue after that age than before it. To produce cell- 
division it is necessary for a cell to absorb a certain 
quantity of an auxetic, and to produce augmented 
asymmetrical divisions by an alkaloid it requires a 
certain combination (as already specified) between the 
alkaloid and the auxetic. If cancer is due to this 
combination, it is possible that before the age of 40 
there is not usually sufficient free auxetic to produce 



SITE-INCIDENCE 363 

the right combination, for we have shown that alkaloids 
by themselves are not effectual in inducing cell-division. 
On the other hand, after the age of 40 the slight 
physiological increase in the quantity of auxetics 
present in a tissue, owing to excess of products of 
katabolism, may just supply the required quantity of 
auxetic to produce the right combination between 
them and an alkaloid, should the latter be present. 
It has already been suggested that the onset of 
cancer may be partly due to the oversetting of a 
normal balance. 

In connection with this point we would recall the 
fact that cancer seems to attack persons who are 
prematurely aged, especially those who are subject to 
such diseases as the atrophic form of osteoarthritis — 
a fact which seems to bear out this explanation of the 
age-incidence of cancer. 

Conversely, it has been shown that cells with a 
lowered vitality require more of an auxetic to produce 
cell-division in a given time than normal cells; and 
this may explain why cancer does not so commonly 
occur in the aged and infirm, for although the right 
combination of the cause of the disease may be present, 
it is not present in sufficient strength to produce 
malignant proliferation in cells which have lost their 
vitality to some extent. 

The suggestion that cancer may be due to putre- 
factive decomposition of the remains of dead tissues 
in a chronic healing site will harmonise with the fact 
that cancer occurs commonly in certain sites. Carci- 
noma occurs most frequently in the breast, uterus, 



364 THE PROLIFERATION OF CANCER 

mouth, stomach, intestinal tract, and rectum. A 
chronic healing focus in the rectum, mouth, or intestine 
may readily be associated with decomposition products. 
As already pointed out, chronic irritation means chronic 
cell-proliferation of healing due to the auxetics con- 
tained in dead cells, and in the rectum, intestine, and 
mouth further decomposition with the gradual produc- 
tion of alkaloid must easily occur in a chronically 
injured site in these regions. It is interesting to note 
that cancer of the pancreas nearly always attacks the 
44 head" of that gland, namely, that part of it which has 
nearest access to the intestine. In the rectum "irrita- 
tion" must be of frequent occurrence by the impaction 
of faeces, and this in itself will obviously render this part 
of the alimentary canal a common site for malignant 
disease — and it is one of the commonest places for it. 
In the mouth decomposition readily occurs, and how 
commonly one sees carcinoma of the tongue, lips, 
fauces, etc. 

Syphilis is undoubtedly a predisposing factor. Syphil- 
itic lesions of the mouth, which, of course, are ac- 
companied by healing, are of very common occur- 
rence, and it is possible that choline is produced in 
tertiary syphilides. If choline is produced by the action 
of Trypanema pallida, then the cause of syphilis may 
be a predisposing cause of cancer — that is, if the 
argument is correct that the alkaloid choline in cer- 
tain combination with auxetics gives rise to malignant 
proliferation. 

The breast and cervix uteri are localities which are 
very prone to cancer, and in these organs destruction 



PRODUCTION OF ALKALOID 365 

of tissues occurs to some extent every month until 
the climacteric, when great involution takes place. It 
is during this latter period that the onset of carcinoma 
is favoured. It is a remarkable thing that cancer 
almost only occurs in these parts in parous women, 
whereas in nulliparous women they are comparatively 
free. We do not think we are going too far in suggest- 
ing that in parous women, when the ducts of the glands of 
the breast and uterus have been — so to speak — opened 
up, access is now afforded to the organisms of decom- 
position and putrefaction. In nulliparous women, when 
these organs have remained functionless, the ducts of 
their glands are more likely to be closed to invasion 
from without. 

In any site, however, the products of katabolism 
may determine the age-incidence of carcinoma. 

One cannot assert that the alkaloid choline, or 
cadaverine either, are only produced in a damaged 
site by the action of putrefactive organisms.. It was 
owing to decomposition by putrefaction of extracts 
of dead tissues and giobin that we were enabled to 
obtain augmented asymmetrical cell-divisions with these 
alkaloids, but it is possible that these alkaloids — or 
others equally effective — may be produced by other 
agencies; and if so, provided the contention is correct 
that the alkaloids help to cause carcinoma, other 
agencies besides putrefactive organisms may cause the 
disease. The point is an important one in view of 
the controversy as to whether cancer is a "parasitic" 
disease. In any case these alkaloids can be produced 
by more than one class of organisms, and we have 



366 THE PROLIFERATION OF CANCER 

pointed out that the cause of syphilis may also 
produce one of them — in fact, General Paralysis of 
the Insane has been said to be due to choline. 
Hence, if our contention is correct, cancer can hardly 
be said to be due to a specific parasite. 

The mere fact that the alkaloids were being pro- 
duced in a chronic healing site would not necessarily 
cause in it augmented proliferation. The alkaloids 
and the auxetics will have to be present in certain 
proportion; and since the production of this pro- 
liferation necessitates the diffusion of the combination 
into the cells, time is an essential factor. In all prob- 
ability it would be necessary for the decomposed 
remains of dead tissues to be pent up to some extent 
for a considerable period. 

The suggestion that cancer may be due to the 
combination of auxetics and ptomaines will offer an 
explanation of the cause of death from the disease. 
If the malignant cells and healing site are completely 
removed, the patient may recover; but if this is not 
the case, recurrence will, of course, take place, for in 
removing the growth a fresh healing site is produced, 
and the original decomposition may go on in it. 
Putrefaction of the remains of dead tissues may occur 
in a healing site without visible suppuration. The 
Bacillus subtilis does not produce pus, yet it will 
produce choline. It may be these ptomaines which 
ultimately cause the death of the patients by poisoning 
them; for if decomposition sets in in a damaged site, 
unless steps are taken to remove it, doubtless the 
decomposition will usually go on. 



METASTASIS 367 

The possibility of carcinoma being due to the 
combination of alkaloids and auxetics will also 
explain the reason for the way in which malignant 
growths frequently "break down." As shown by 
in-vitro experimentation, cells can only withstand a 
certain quantity of the combination. If excess is 
forced into them, they will die. Even globin itself 
is very poisonous to leucocytes and lymphocytes if it 
is in excess. If this excess was present in the body, 
it would cause cell-death unless the excess was 
removed, and the cell-death would only aggravate 
the trouble, especially if the lymphatics were blocked 
by malignant cells. Ultimately, of course, the growth 
would "point" and break down. A certain amount 
of local cell-death will cause increased cell-proliferation ; 
but after a certain stage is reached, breaking down must 
occur with subsequent ulceration. 

Up to a certain point, therefore, the greater the 
malignant proliferation, the more cell-death will there 
be, and the more will the disease be aggravated. 
And the aggravation may be increased by the 
chromosome granules which cells appear to discard 
when they are excessively prolific. These chromatin 
granules may contain kreatin, and they will therefore 
merely supply more auxetic for the neighbouring 
cells. 

The phenomenon of metastasis in cancer is an im- 
portant factor to be considered in conjunction with the 
other facts. The invasion of lymphatics by cancer may 
be due to the combination of auxetics and alkaloid 
being passed through them from the original healing 



368 THE PROLIFERATION OF CANCER 

site. We have seen amoeboid movements in cancer- 
cells in response to alkaloids, and possibly this may 
assist in the infiltration of vessels and tissues and so 
predispose to metastasis. A striking fact known about 
secondary growths is that in the arrangement of the 
cells they resemble the primary ones. We think that 
this can be explained only by embolism. If a second- 
ary growth in another organ was a fresh cancer, it 
is difficult to imagine how it could possibly resemble 
the primary ones in the arrangement of the cells. 
Metastases practically only occur in the later stages of 
carcinoma when the lymphatics have been extensively 
invaded. In benign growths one rarely if ever sees the 
vessels invaded by cells, and presumably this is the 
reason why secondary tumours do not follow. The 
extensive researches which have been done by others 
in transplanting tumours in mice have thrown con- 
siderable light on the nature of secondary growths. 
In transplanting a tumour from one animal to another, 
it seems to us that one is in reality producing a secondary 
tumour. Now, to effect this, as is well known, it is 
necessary that the cells of the tumour should be alive; 
the transplanting of dead cells will not cause a second- 
ary growth. This knowledge harmonises with our 
suggestions as to the cause of cancer. If one inoculates 
an animal with dead cells, although the organisms of 
putrefaction may be present among them, the remains 
of the dead cells are soon removed from the inoculation 
site and the production of augmented auxetic must 
cease. Normal healing will take place before the 
putrefactive organisms have had time to restart and pro- 



METASTASIS 369 

duce choline, cadaverine, etc., for it is known 1 that to 
produce these alkaloids it takes at least a fortnight. 
If, on the other hand, a portion of a living primary 
growth is transplanted, the living cells will continue 
to multiply in response to the auxetics produced by the 
cell-death which continues to occur among the malig- 
nant cells which have been inoculated. In transplanting 
a malignant growth, one must transplant some putrefac- 
tive organisms along with the malignant cells, and in 
the spaces between the cells the combination of auxetics 
and alkaloids must be present from the outset and be 
continuously produced without interruption, because a 
living growth is transplanted. For a secondary growth 
(or a metastatic one) to occur, it is necessary for living 
cells to be transplanted; and we believe that it is also 
necessary for organisms to be transplanted within it, so 
that the causes of the augmented proliferation continue 
to be supplied without interruption. 

There is another possible explanation of a metastatic 
growth which should be mentioned. It has been sug- 
gested by others, who, of course, were unaware that 
cell-division in the body is caused by chemical agents, 
that once a cell becomes a malignant one, its daughter 
cells will also be malignant. This would mean that 
a cell, in acquiring malignant characteristics, would 
transmit those characteristics to its progeny. This 
would be a "mutation" — an acquired characteristic 
suddenly becoming hereditary for all succeeding genera- 
tions; an event which we think is most unlikely to 

*It must be remembered that these organisms may have nothing to do 
with either sepsis or suppuration. 

24 



370 THE PROLIFERATION OF CANCER 

occur. It is difficult to imagine how a cell, having 
started augmented divisions in response to a combina- 
tion of alkaloid and auxetics, could in its subsequent 
generations continue to divide by augmented divisions 
when the cause of the augmentation is absent. We 
have shown experimentally that if the supply of auxetic 
to a cell ceases, the cell-division also ceases. This 
experiment tends to dispose of the expressions "first 
(heterotype) divisions and subsequent (homotype) 
divisions," which in reality imply a mutation. We 
think, therefore, that a metastatic growth consists of 
a portion of the primary one transplanted elsewhere 
along with some of the original cause of its augmented 
proliferation. 

It is possible that in the later stages of cancer the 
body-fluids may contain considerable amounts of alka- 
loid, derived from the primary growths, which might, in 
the event of a fresh healing focus occurring anywhere, 
be sufficient to act in combination with the new local 
auxetics, and so cause another "primary" growth. If 
such occurred, it would probably be mistaken for and 
called a secondary growth. 

Lastly it may be mentioned that if cancer is due to 
putrefaction occurring in a chronic healing site, there 
may be something in the view upheld by many, that 
the disease occurs frequently in certain localities or 
even in certain houses. Doubtless putrefaction will 
occur more readily in certain places, because the 
bacteria of putrefaction may infest the air there. In 
connection with this I may recall the remark — al- 
ready noted — which was made to me by Sir William 



EXPERIMENT REQUIRED 371 

MacGregor, that he had never seen a case of cancer 
among the Esquimo. 

The " error of random sampling," however, must 
be considered with the question of the "local inci- 
dence" of cancer. Very large figures would have to 
be studied before one could say conclusively whether 
the incidence of the disease is actually greater in some 
localities than in others, and experimentation with 
animals in the confines of the laboratory cannot, we 
think, determine whether putrefaction is more likely 
to occur in one place than in another. Still, the 
remark of Sir William MacGregor is striking, because 
it is clear that putrefactive bacteria cannot be present 
to so great an extent in the Arctic regions as in 
temperate and tropical climates. 

The above consideration led us to believe that our 
researches did harmonise with the facts known about 
carcinoma. The fact that cell-proliferation is caused 
by auxetics contained in the soluble remains of dead 
tissues offers for the first time an explanation of 
the remarkable age-incidence of the disease; and the 
augmented asymmetrical division induced by these 
auxetics combined with alkaloids of putrefaction 
seemed to be a reasonable explanation of the cause 
of cancer. Proof was wanting, however. Cancer-cells 
have been seen frequently to divide by asymmetrical 
divisions, but because one can induce these mitoses in 
cells is not proof that one is necessarily inducing 
malignant proliferation. 1 

1 As a matter of fact, the five-fold augmentation by alkaloids is a more 
important consideration than the asymmetrical mitoses induced by them. 



372 THE PROLIFERATION OF CANCER 

Deductions from experimentation in vitro, no 
matter how well they may harmonise with known 
facts, are not sufficient to act as a basis on which to 
proceed to find the prevention and cure for the disease. 
It is necessary at least to try to prove one's work 
definitely. To accomplish this would not be, we knew, 
an easy matter. It would be necessary to produce 
a cancerous growth in healthy animals with the sub- 
stances which were believed to be the cause of the 
disease. The chemical auxetics, in correct combination 
with an alkaloid of putrefaction such as choline, would 
have to be inoculated into or applied to an animal, and 
before one could say that the combination is a cause of 
cancer a malignant growth would have to appear at the 
site of inoculation. The experiment would have to be 
frequently repeated, and careful precautions would have 
to be taken against possible fallacy. 

It was realised that it would be quite useless merely 
to inoculate a solution of, say, kreatin and choline sub- 
cutaneously into an experimental animal, because it is 
obvious that such a solution would rapidly be excreted, 
and we know from in-vitro experimentation that before 
a cell can divide, either by a normal or an asymmetrical 
division, it must be subjected to the chemical agent for 
a certain length of time. It would be necessary to 
create a sore, because a chronic healing site is essential ; 
and this would not be readily accomplished in experi- 
mental animals, which are not easy to keep quiet, and 
in which the local application of substances to sores 
offers practical difficulties. 



EXPERIMENT REQUIRED 373 

Moreover, the question whether the lower animals 
suffer from true cancer is still controversial. I 
therefore considered whether it would be possible to 
try this crucial experiment on a human being. If it 
were possible, and if it were successful, the point might 
be proved conclusively. At first sight the suggestion 
seems to be an outrageous one, but the experiments to 
be related in the next and last chapter, which had been 
carried out for several months past, revealed a method 
by which I considered that an attempt might be made 
to put this crucial experiment to the test. 



CHAPTER XVII 

INHIBITORY ACTION OF BLOOD-SERUM ON AUXETICS 

MEASUREMENT OF THIS ACTION THE TREATMENT 

OF SOME CASES OF CANCER BY THE ADMINISTRA- 
TION OF DEFIBRINATED BLOOD DESCRIPTION OF 

THE CASES THE TREATMENT OF A MALIGNANT 

ULCER BY MEANS OF GLOBIN AN ATTEMPT TO 

MAKE THE CRUCIAL EXPERIMENT CONCLUSION 

It is now (August, 1910) more than six months since 
it was ascertained that leucocytes and lymphocytes 
divide in response to the auxetics contained in the 
remains of dead tissues and in globin. When this 
fact was appreciated, the question arose as to why 
these cells, when they are removed from the peripheral 
circulation, had never been seen in the act of cell- 
division. White blood-corpuscles were discovered by 
Hewson in 1773; in 1846 Wharton Jones first described 
them as granular and nucleated cells (Buchanan). 
Since then they must have been seen by every student 
of medicine, but no one, until divisions were induced 
in them by us, had ever seen one of these cells divide. 

374 



NO MITOSIS IN BLOOD STREAM 375 

Hence it is obvious that these cells do not divide in 
the peripheral circulation, for their mitosis occupies 
a certain amount of time; and if this mitosis occurred 
at all in the peripheral blood, it must have been seen 
during the century and a half in which these cells have 
been constantly examined by many thousands of workers. 
Now, the division of these cells is caused by the 
auxetics contained in the remains of dead tissues and 
in globin, and it also is certain that the peripheral 
blood must contain some free remains of dead tissues 
and globin. Hence white blood-corpuscles ought to 
be frequently seen in the act of division when they are 
removed from it. But they are not so seen. Had it 
been seen, the real nature of the Altmann's granules 
and the "lobes of the nuclei" would have been apparent 
many years ago. 

We think that there can be only one explanation 
for this, which is that the action of the auxetics in the 
peripheral blood is restrained in some way. It appears 
to us to be reasonable to suppose that cell-proliferation 
in the peripheral circulation must be prevented in some 
way. If it were not, the approach of old age or a 
chronic suppurative focus with destruction of tissue 
might cause indiscriminate cell-proliferation in the 
vessels and capillaries, with disastrous results ; for these 
vessels might ultimately become blocked. We there- 
fore made some experiments to see if blood-serum does 
actually restrain cell-division. 

In the first place, 2 cc. of sheep's serum was added 
to auxetic jelly composed of azur dye, atropine, and 
suprarenal extract. In order to prevent coagulation of 



376 PREVENTION OF PROLIFERATION 

the serum in this and the subsequent experiments, the 
serum was added to the jelly after the latter was boiled 
and before it had cooled to such an extent as to prevent 
it setting on the slide. It was found that the serum 
did not prevent the cell-division induced by the azur 
stain. 

The experiments were then repeated with a jelly 
which contained suprarenal extract, but no stain or 
atropine. The jelly was first tested, and mitotic figures 
induced in lymphocytes with it. The jelly contained 
0.2 gramme of suprarenal extract, and it was found 
that if it also contained 0.5 cc. of serum the auxetic 
action of the extract was not stopped; but if it con- 
tained 2 cc. of serum the auxetic action of the 
suprarenal extract was completely inhibited. 

The experiments were then repeated with an auxetic 
jelly composed of a mixture of 1 cc. of a 1-per-cent 
solution of kreatin and 1 cc. of a 1-per-cent solution 
of choline; and it also contained 10 units of alkali 
solution. With this jelly it required the addition of 
2.5 cc. of sheep's serum to prevent it causing cell- 
division. 

Using human serum, it required 2 cc. of it to stop 
the action of . 2 gramme of suprarenal extract by itself. 
1 cc. of serum will stop the action of the combination of 
0.01 gramme of kreatin and 0.01 gramme of choline; 
1 cc. of human serum will stop the action of . 5 cc. of a 
2-per-cent solution of globin which had been allowed 
to become putrid, and which would, by itself, induce 
division in lymphocytes. 

Hence it is apparent that normal blood-serum 



CELL-DIVISIOX RESTRAINED 377 

actually has the power of preventing the "natural" 
auxetics from inducing cell-division; but it has no 
inhibitory action against atropine or azur dye. The 
restraining power of serum can be measured as shown, 
and it is possible that this power varies with individuals, 
a point which remains to be determined. 

It was also ascertained that the restraining body 
in serum does not combine permanently with the 
auxetic and so prevent its action. Jellies were pre- 
pared with suprarenal extract with kreatin and choline, 
which induced divisions in lymphocytes. The right 
amount of serum was added to them just before the 
jellies cooled, and it was noted that they stopped the 
auxetic action of the jellies. The same jellies were 
then boiled and the serum proteins precipitated. On 
making specimens again from these jellies, it was now 
found that their auxetic power was re-established. 
Hence it is obvious that the restraining body in serum 
is not thermostable. 

Lastly, it was found out that 1 cc. of serum con- 
tained in 10 cc. of jelly which also contained 1 cc. of a 
1-per-cent solution of choline stopped the kinetic ac- 
tion of the latter in exciting amoeboid movements in 
leucocytes. If the jelly was boiled, however, the action 
of the choline was restored. 

These experiments are very constant in their results. 
Careful controls were made throughout. We think 
that by means of them the restraining power of 
different sera could be measured with a certain amount 
of accuracy. What the nature of the restraining body 
in serum is we have no opinion to offer. It should be 



378 PREVENTION OF PROLIFERATION 

noted that some time ago Bashford and Murray showed 
that serum had the power of restraining the growth of 
secondary transplanted tumours in mice. 

In addition to the restraining action of serum 
on the cause of cell-division, we also considered 
the work of Gaylord and Clowes of the New York 
State Cancer Research, Buffalo, and of Bashford and 
his assistants at the Imperial Cancer Research in 
London, who have shown experimentally that the 
transplantation of living growths in mice protect them 
to some extent against cancer. It was considered 
possible that this might be due to the fresh augmented 
auxetic produced by the transplanted growths giving 
rise to an increase in the content of the restraining 
body. We therefore resolved to try to increase this 
body in cancer patients by deliberately injecting them 
with augmented auxetic combined with blood-serum. 
The way the combination was administered was by 
injecting 6 ounces of defibrinated sheep's blood per 
rectum every morning. The serum contains the re- 
straining body, and it was argued that the red cells 
would be destroyed in the rectum, the haemoglobin 
decomposed, and in time the globin would become 
augmented by the action of the bacteria present. It 
was presumed that the restraining body of the serum, 
the auxetic in the globin and in the remains of the 
white cells, and lastly, the products of the decompo- 
sition would be gradually absorbed, and that they might 
raise the content of restraining body in the patients; in 
other words they might act as a sort of vaccine. 

We must admit that we were not very sanguine of 



CLINICAL CASES 379 

success when these experiments were first undertaken 
six months ago. They were undertaken more with a 
view to see what the effect of globin in this way was 
than with the object of obtaining a cure of the tumours 
from which the patients were suffering. But, as will 
be seen from the description of the treatment, the 
results have exceeded our anticipations. Unfortunately, 
since we did not expect any beneficial results, the cases 
were not the most suitable which could have been 
chosen, for both of them had "internal" growths which 
were inaccessible, and therefore we were at that time 
unable to prove conclusively that they were suffering 
from carcinoma. 

The first patient 1 to whom the serum was adminis- 
tered was a woman (I. G.) aged 45 (admitted to the 
hospital on January 11, 1910), whose left breast had 
been removed in November, 1907, for a carcinomatous 
growth. She remained well until April, 1909, when she 
began to suffer from a severe pain in the region of the 
sacrum and left hip. She stated that this pain had 
since then become worse and that no treatment had 
relieved it. The left lower extremity from the hip to 
the ankle had for long been swollen and cedematous, 
and there had been swelling also in the abdomen. 
Any movement of the limb caused severe pain, and 
she had great difficulty in turning herself in 
bed. The patient was too ill to be weighed at the 
time of her admission, but she was manifestly wasted, 

1 These two cases were treated under the supervision of Dr. Macalister, 
who has kindly written these descriptions of them. 



380 PREVENTION OF PROLIFERATION 

was anaemic, and had a worn expression. On exami- 
nation there was manifest swelling on palpation in 
the left iliac region, and, examined per rectum under 
chloroform, a hard swelling could be felt attached to 
the anterior surface of the sacrum and sweeping round 
the wall of the pelvis towards the left side. An X-ray 
photograph confirmed the involvement of the sacrum. 
After treating her with mercurial inunctions and other 
remedies for a month without benefit, the defibrinated- 
blood injections were commenced on February 21, 1910. 
At first they were given in the evening, and were often 
followed by sickness and sometimes by actual vomiting ; 
it was found that there was less disturbance when the 
injections were given in the mornings. 1 The sickness 
was so troublesome at first that the treatment had to 
be abandoned on March 3, and it was not until 
March 20 that it was again started and continued 
uninterruptedly. Gradually her pains improved, and 
the swelling in the leg diminished. (No opiates were 
needed after March 29.) On April 20 she could stand 
with very little pain, and she was weighed for the first 
time (109 lb.). Improvement continued week by 
week, she became bright and younger-looking, and 
on June 8 she weighed 115 lb. No pain could be 
elicited on pressure over the left iliac region, and the 
tumour seemed smaller. She maintained her weight, 
with some variation, for some weeks, and was able to 
walk about the ward without assistance until July 21, 

1 In these cases treated with rectal injections of defibrinated blood 
there has been sickness following the injection, but this has passed off 
as the treatment has been persevered with. 



CLINICAL CASES 381 

when she sprained her left shoulder and suffered severe 
pain in it. On July 19, by the patient's request, 
the treatment was discontinued, and an opportunity 
thus arose of observing whether the benefit which had 
resulted from it was maintained. There had been some 
sciatica-like pains in the leg since the beginning of the 
month, and during August these increased and the 
swelling and pain in the hip returned. Some tender- 
ness and a tumour, apparently arising from the medias- 
tinum and which grew rather rapidly, appeared in the 
mid-sternal region. By the first week in September, 
she had relapsed pretty much into the condition in 
which we found her at the time of her admission, but 
with the added pain due to the thoracic growth. The 
treatment has now been resumed. 

The second case was that of a woman aged 54, 
who had suffered from indigestion for a considerable 
period, but severely for three months. There had been 
much vomiting, but never any blood. At the time of 
her admission (February 9, 1911) ingestion of food 
was immediately followed by severe pain, and often 
by sickness. She was very wasted, worn-looking, 
and anaemic. Weight 94 lb. On examining the ab- 
domen a swelling could be seen and felt above the 
umbilicus. It was about the size of a tangerine orange. 
It was extremely tender, and moved with respiration. 
The stomach was very dilated, and presented peris- 
taltic movements. There was pain on pressure over 
the pyloric region, but no tumour could be felt there. 
The stools contained altered blood (meloena). During 
the first fortnight after admission, when she had milk 



382 PREVENTION OF PROLIFERATION 

and Benger's Food, the vomiting ceased, and she had 
less pain, but she lost four pounds in weight (90 lb.). 
The defibrinated blood was commenced on February 21, 
and until March 23 the weight fluctuated between 
88 lb. and 91 lb., there being an occasional gain 
and then a corresponding loss; but on March 30 
a steady advance commenced, the maximum weight 
being attained on May 8, when it reached 101 lb., 
i.e. a gain of eleven pounds since the time of her 
admission. From the time of the commencement of 
the defibrinated-blood treatment she steadily improved. 
She became able to eat fish and a light ordinary diet 
without discomfort; but the most striking fact was 
the diminution in size of the tumour, which practically 
disappeared. As in the former case, after reaching a 
climax there was a recrudescence of the symptoms, 
and some loss in weight, but the tumour did not 
return. The defibrinated blood was omitted on July 
20, when she weighed 95 lb. Subsequently she 
mended, and left the hospital on August 9 consider- 
ably better and weighing 100 lb. There was un- 
doubtedly some real improvement in this case, and 
the temporary relapse depended to some extent on 
fermentative changes taking place in the dilated 
stomach. 

Several other cases have been treated with the 
defibrinated blood, and in some of them there has 
been apparent benefit, although others (a case of very 
advanced cancer of the liver, and one of peritoneal 
cancer) have not shown improvement. 

In addition to the rapid reduction in size of the 



CLINICAL CASES 383 

growths in these two cases, a striking point was the 
improvement in the general symptoms and appearance 
of the two patients. Their cachexia? practically dis- 
appeared, they became cheerful and seemed to get 
younger. In the first case the disappearance of the 
oedema of the legs was most remarkable, and never 
before had we seen cases of carcinoma, which had been 
bed-ridden for months previously and condemned by 
surgeons as being inoperable, become able to be up 
and about apparently vastly improved in health. It 
must be distinctly understood, however, that we do 
not assert that this treatment is in any way a cure 
for the disease. As mentioned at the outset of the 
description of the cases, we have no absolute proof 
that they were cases of carcinoma, and it must be 
remembered that spontaneous improvement and cure 
in some cases of cancer have undoubtedly occurred 
without any ^treatment whatever. Gaylord and Clowes 
have collected a series of these cases. 1 Moreover, 
we have been able to deal with only a few cases, and 
they have been under observation for only six months; 
therefore we cannot say whether the results are going 
to be permanent or even maintained for any length of 
time. 

The reason why these cases are described is that they 
suggested to me a possible way in which the crucial 
experiment, mentioned at the end of the last chapter, 
could be carried out. It appeared reasonable that if 
one can cause the reduction in the size of a growth with 

1 Seventh Annual Report (Cancer Laboratory, New York State Depart- 
ment of Health). 



384 PREVENTION OF PROLIFERATION 

amelioration of symptoms by general treatment, one 
might also be able to improve an accessible growth by 
locally inducing the proliferation of healing in it. If 
this were possible, and if a local, inoperable, broken- 
down scirrhus could be so improved by local treatment as 
to replace some of the infiltrating cells by normal ones, 
I considered that I should be justified then in carry- 
ing out the crucial test on these normal cells, and try to 
reinduce the abnormal infiltration amongst them once 
more by the direct application of auxetics and choline. 
In other words, if a case already has a large "inopera- 
ble" tumor and one is able to convert by treatment a 
portion of it into normal tissue, it would be useful to try 
temporarily to reconvert the normal tissue back into 
original condition in order to prove the main point of 
our researches. There would already be a neoplasm, 
and I proposed thus to test our theory in in vivo on a 
portion of it. 

A patient suffered from an inoperable, f ungating 
scirrhus of the breast. The ulcerated surface was about 
four inches in diameter. The edges were precipitous 
and excavated, and the whole appearance of the ulcer 
was typical of carcinoma. The surface was practically 
devoid of granulation tissue, and sections of it clearly 
showed its nature (figs. 119, 120). A portion of the sur- 
face, i.e. about a third of it, was scarified and globin 
was applied by being "dotted" over it (fig. 121). The 
remaining part of the ulcerated surface was untreated. 
No dressings were applied. This ulcer had a remarka- 
ble propensity for suppurating. No matter what was 



LOCAL EFFECTS OF GLOBIN 



385 





L 




Fig. 119. — Section from the case of scirrhus of the breast. Low power. 




Fig. 120. — The same as 119. High power. 



LOCAL EFFECTS OF GLOBIX 



387 




Fig. 121. — To show the way in which globin wai 
of the malignant ulcer 



dotted" on to a portion 



LOCAL EFFECTS OF GLOBIN 389 

done to try to keep it "clean," pus quickly formed on 
its surface — much more quickly than it did on the be- 
nign callous ulcers which were also being treated. The 
result was that the scab formed by the globin very 
quickly broke down. When this occurred, the scab was 
removed by fomentations and the ulcer cleaned up as 
much as possible. Then the globin treatment was re- 
peated, but it was always applied to the same portion 
of the ulcer. The other portion never received an ap- 
plication. This was repeated many times. 

The improvement in the treated portion was gradual, 
but it was marked. The precipitous edges appeared 
to soften and become flattened. The base no longer 
suppurated in a few hours, and the suppuration was 
practically confined to the untreated portion. The 
glistening malignant surface of the treated portion 
gradually gave place to granulation tissue, and after 
about a fortnight's treatment there was a contrast be- 
tween the treated and untreated portions of the broken- 
down surface. A portion of the treated part of the 
ulcer was now removed and sections cut from it, which 
show that the abnormal cells were now giving place to 
normal granulation tissue. 

The treatment was continued once more, two parts 
of kreatin now being added to five parts of globin, 
and soon it was seen that the treated portion became 
softer, and the ulcerated edge ceased to extend. 
Another section was then cut, which showed that 
that part of the ulcer now seemed to be devoid of 
abnormal infiltration (figs. 122, 123). 



390 PREVENTION OF PROLIFERATION 

I considered that the opportunity had now ar- 
rived to try the crucial test. A mixture was made 
of a solution containing five parts of globin and 
one part of choline. It was evaporated to dryness 
with aseptic precautions, and the dried mixture sealed 
up in a glass tube. A minute portion of the edge of 
the treated ulcer, from which the last section had been 
taken, was now scarified and small pieces of the 
dried aseptic mixture of globin and choline directly 
applied to it. In 48 hours a conical excrescence 
appeared at the seat of application. A section has 
been cut from it, and the photomicrograph shows 
apparently new malignant cells to be infiltrating the 
granulation tissue once more in an abnormal manner 
similar in appearance to the original infiltration of the 
ulcer (figs. 124, 125). 

Now, this test is by no means conclusive. I can- 
not assert that there were no original carcinomatous 
cells at the seat of application, and that I was 
not merely producing augmented proliferation of 
these cancer-cells. The section may be fallacious 
owing to the "error of random sampling," for be- 
cause no abnormal cells appear in samples removed 
from the treated site it does not prove that none 
exist in the neighbourhood. Still, the experiment is 
interesting, because the conical excrescence only 
appeared at the site of the application of the com- 
bined auxetie and the alkaloid of putrefaction, the 
rest of the ulcer remaining in statu quo. 

This test will have to be repeated many times 
before one can speak conclusively on the subject; 



r 



ATTEMPTED CRUCIAL TEST 



: i > 



391 




r . v 







7>' 







Fig. 122. — Section of a portion of the ulcer after treatment. Low power. 



f- 



-' 



k 






Fig. 123. — The same as 122. High power. 



ATTEMPTED CRUCIAL TEST 



393 




■MHHH^B 



-1 



L 




Fig. 124.— Section of the treated portion of the ulcer after the application 
of globin augmented by choline, showing reinfiltration. Low power. 




Fig. 125. — The same as 124. High power. 



ATTEMPTED CRUCIAL TEST 395 

but from the general appearance of the ulcer, 
as well as from the microscopical section of it, we 
certainly think that it was the mixture of globin 
and choline which caused the reinfiltration. The 
application of globin alone to the edges of the 
sore merely induced the appearance of granulation 
tissue, and one would think that if a mixture of 
globin and choline does not cause carcinoma, it 
would merely have produced augmented proliferation 
of the healing cells with more granulation tissue; 
but apparently a new infiltration of epithelial cells 
appeared. I hope to be able to repeat this test 
under more favourable conditions. 

The whole ulcer is now being treated with a 
mixture of globin and kreatin, and, although the 
edges of it are extending in some places, there can 
be no doubt that, on the surface at least, malignant 
cells are being replaced by normal granulation tissue. 
The Avhole growth is now comparatively freely mov- 
able, and it does not discharge profusely as it did. 
The patient no longer complains of pain, and, except 
for the extending edges, her general condition has 
greatly improved. 

As it is possible that carcinoma may be due to 
the causes described in this book, and since the 
general treatment by defibrinated blood per rectum 
and the local treatment by globin and kreatin seem 
to have been followed by improvement, we think 
that the former might be tried to prevent recurrence 
after removal of a growth. Unfortunately we are 
not in a position to try this experiment, as early 



396 PREVENTION OF PROLIFERATION 

cases which have been operated on do not come to 
our notice; we therefore take the liberty of suggesting 
that the prevention of recurrence might be under- 
taken by those who can watch a series of these cases 
which have been operated on. We cannot say, of 
course, whether recurrence will be prevented by rectal 
injections of defibrinated blood, but the treatment 
is harmless and it appears to be worthy of a trial. 

If cancer is due to the causes which we think it to 
be, the reason for recurrence after removal of the 
original growth, which occurs in some cases in the 
operation scar, is open to two explanations: (1) that it 
may be due to portions of the original growth which 
have not been removed, and (2) that the healing site, 
although healing occurs by first intention, is a fruitful 
source of auxetics, and that the operation wound may 
easily become infected by the putrefactive organisms 
during the removal of the original growths. As already 
pointed out, certain putrefactive bacteria do not neces- 
sarily also cause suppuration; and therefore recurrence 
in the scar may be due to a fresh attack of cancer there. 
The proliferation of healing (even in a site healing by 
first intention) probably continues for weeks if not for 
months after the injury, because the initial proliferation 
increases the number of deaths, and possibly it is a long 
time before normal elimination is sufficiently restored to 
put a stop to the abnormal proliferation. 

One frequently hears of cases in which "recurrence" 
takes place perhaps ten years after removal of the 
original growth. This must be due to a fresh attack of 
cancer. One of the commonest sites for it is in the 



THE RECURRENCE OF CANCER 397 

rectum, the very place where one would expect it to 
occur. Now that we know the causes of cell-prolifera- 
tion it is difficult if not impossible to believe that a 
metastatic growth could remain malignant and quiescent 
for ten years without proliferating. We think that the 
increase of "restraining body" conferred on a person by 
a malignant growth may not last very long, and this, 
coupled with advancement of old age and possibly the 
existence of physiological excess of general auxetics 
which may occur in some persons, may predispose them 
to subsequent attacks of cancer. Our argument is that 
cancer is due to a combination of physiological auxetics 
and pathological alkaloids of putrefaction. The combi- 
nation must be a definite one, or it will not be effectual ; 
it must diffuse into the cells to a certain extent for a 
certain length of time, with due regard to the coefficient 
of diffusion of the cells; and lastly the vitality of the 
cells themselves must not be greatly impaired. We 
think that unless all these factors are in correct combina- 
tion, malignant disease cannot occur. 

With regard to the cause of sarcoma, we think that 
it is probable that the auxetic chiefly concerned in that 
disease is that contained in globin. Several surgeons 
have kindly informed -us that in almost every case of 
sarcoma which they have seen there is a history of 
injury; and it is remarkable that sarcoma occurs most 
frequently in those tissues which are rich in haemo- 
globin, namely, the choroid coat of the eye (melanotic 
sarcoma), the bone marrow, and the neighbourhood of 
muscles. The suggestion that globin is the source 
of the auxetic in sarcoma will explain the age-incidence- 



398 PREVENTION OF PROLIFERATION 

of the disease; for it probably only follows injury to 
large numbers of red cells. The length of life of red 
cells in the body is supposed to be only a matter of a 
few weeks, so that their anabolism and katabolism is 
continuous, and may not depend at all on the age of the 
person. Hence sarcoma may occur at any age. 

Whether the alkaloids of putrefaction are concerned 
in sarcoma or not, we are not in a position to state, but 
interesting cases have been reported from time to time 
which were associated with suppurative foci. Quite 
recently a case was described in The British Medical 
Journal, 1 of an infant which had been injured in the 
neck by forceps at birth. Sarcoma followed on the 
injury, which was also complicated by suppurative otitis 
media. 

The possibility of the alkaloids in both sarcoma and 
carcinoma being of the nature of leucomaines which are 
supposed to be absorbed from the intestines must not 
be forgotten. 

The proliferation of leucocytes and lymphocytes 
in the leukaemias are also doubtless due to auxetics. 
Whether these diseases are caused primarily by injury 
to the spleen or not we do not know, but it is possible 
that this starts the proliferation. The spleen tissue has 
direct access, by means of the vessels, with the per- 
ipheral circulation, and presumably this is the reason 
for the leucocytosis and lymphocytosis in leukaemia. 
It is impossible to say whether the proliferation of 
leukaemia is of the augmented type, or whether an 

1 " A Case of Sarcoma of the Pectrous Bone," by W. H. Bowen and H. B. 
Carlyle (B.M.J., June 25, 1910.) 



CONCLUSION 399 

alkaloid is present; but we may recall the interesting 
fact mentioned by Buchanan in his admirable book on 
the clinical pathology of the blood, 1 that he had noticed 
the discard of granules (flagellation) in the cells of cases 
of leukaemia. Possibly the leukaemias may be associ- 
ated with the auxetic contained in globin, for the spleen 
is a very vascular organ; and if so, it may ultimately 
be found that leukaemia is a from of sarcoma of the 
spleen. 

In concluding these descriptions of the researches 
which we have been able to carry out to the end of 
the first year and a half of the establishment of the 
Research Department of the Royal Southern Hospital 
at Liverpool, I wish once more to acknowledge my 
indebtedness to all those who have helped me so 
materially. I think that the new methods at our dis- 
posal have been the means certainly of solving the 
problem of the cause of normal human cell-division, 
and possibly, if not probably, of the cause of malignant 
cell-proliferation also. Much work remains to be done, 
however, some of which has already been started. 

A series of more than ten "inoperable" cases of 
cancer are now being treated by defibrinated blood and 
by the local application of normal auxetics. Experi- 
mentation is begun to ascertain what organisms produce 
substances which "augment" the action of auxetics. 
The strength of the body in normal serum which 
restrains cell-division is being measured with a view 

1 R. J. Buchanan, The Blood in Health and Disease (Oxford Medical 
Publication). 



400 PREVENTION OF PROLIFERATION 

to see if it varies in different persons, both in normal 
and in pathological conditions. The lengths of the lives 
of leucocytes are being measured in the presence of 
various strengths of auxetics and alkaloids of putre- 
faction. Many fields of work are now opened by the 
knowledge that the reproduction and multiplication of 
the cells of our bodies are due to certain known (and 
some as yet unknown) chemical agents. The knowledge 
that " healing" itself is caused by these agents may ulti- 
mately assist the medical man in his work, and I think 
that it will be found that trypanosomes amoebae (the 
causes of dysentery), and other parasites also multiply 
in the body in response to the remains of dead cells. 
These paths of research will require many workers, and 
I am sure that their investigation will not be wasted. 

Whether the benefit derived from the treatment 
adopted in two of the cases of cancer will prove to 
be of practical value or not remains to be seen. In 
any case it is capable of elaboration and further investi- 
gation. Even if it confers the smallest amelioration 
of symptoms, which it undoubtedly appears to have 
done in these two cases, something has been accom- 
plished; but whether the benefit is lasting or not time 
alone will show. 



APPENDIX I 



ENUMERATION OF THE NUMBER OF GRANULES 
EOSINOPHILE LEUCOCYTES 



CONTAINED IN 



TABLE I 1 

Controls (healthy and diseases other than cancer) . Males 



Name. 



Age. 



Disease (or Health). 



G 



9. <» 

^ o ■ 

- ~" .- 

o o 
H 



HI 

:» = 



§ = 5 



a; 
- - u 



Connolly 
Parker . 
Edwin . . 
Brewer . 
Hughes . 
Mattison 
Duncan 
Bradley 
Holding 
McDonald 
Stevens 
Ketch . 
May . . 
Ball . . . 
Armstrong 
Mahoney 
Berry. . . . 
Hankinson 
Cropper . 
McConnell 
Grue . . 
Jones . 
Ross . . . 
Smith . 
Ritchie 
Morgan 
Daulbv 
Noble . . 
Braig . 
Cann . . 
Gould . 
Lowry . 
Benn . 



12 Chorea 

14 Healthy 

15 Healthy 

15 Mitral Disease 

15 Osteo-arthritis 

17 Malaria 

20 Filariasis 

21 Pneumonia 

22 Healthy 

24 Fracture 

24 Sarcoma 

25 Varicocele 

25 Fracture 

25 Pneumonia 

26 Sleeping Sickness . . . 

26 Sarcoma 

27 Floating Kidney 

28 Acute Nephritis 

28 Healthy 

32 Fracture 

34 Hernia 

34 Healthy 

34 Healthy 

36 Fracture 

46 Hernia 

47 Hydrocele 

50 Empyema 

52 Addison's Disease . . . 

60 Stricture 

62 Varicose Ulcer 

65 Healthy 

66 Chronic Rheumatism 
86 Healthy 

Total 



5 


811 


142 


1 


174 




1 


134 




1 


180 




2 


536 


266 


5 


779 


135 


2 


291 


141 j 


4 


561 


124 


2 


354 


177 


1 


227 




2 


368 


169 


1 


155 




3 


738 


204 


3 


502 


101 


1 


82 




1 


175 




2 


475 


197 


5 


798 


125 


21 


3,390 


114 


5 


745 


127 


2 


325 


146 


4 


769 


155 


7 


1,086 


127 


2 


297 


105 


5 


748 


122 


5 


840 


137 


2 


345 


151 


6 


875 


124 ; 


2 


318 


145 


2 


492 


230 


2 


336 


147 


1 


165 




1 


211 


... | 


109 


18,282 





175 



270 
185 
150 

147 
177 

199 

2S7 
220 



278 
200 
260 
165 
179 
208 
190 
192 
192 
206 
194 
190 
173 
262 
189 



Averag 



162 
174 
134 
180 
268 
156 
145 
140 
177 
227 
184 
155 
246 
167 
82 
175 
237 
160 
161 
149 
162 
192 
155 
148 
150 
168 
172 
146 
159 
246 
168 
165 
211 



168 



1 In the averages fractions have been neglected throughout. 

401 26 



402 



APPENDIX I 



TABLE II 



Controls (healthy and diseases other than cancer). Females 



Name. 



Age. 



Disease (or Health) . 



^-o 






X> 03 • 

a^-g 

12a 



sssS 
111 

3 g o3 



RO 



O^ 



as . 

03 ^ ^ 
MO O 

03 O 

■5 



Shankayne 
Frost .... 
Matthews 
Farrington 
Simpson 
Stone . . 
Francis . 
Baker . . 
McKey . 
Jackson 
Harris . . 
Swalwell 
Wilson . 
Benn . . . 



13 J Chorea. . . 

16 J Peritonitis 

17 I Hysteria . 
19 ! Healthy 
21 
22 
24 



Osteo-arthritis 
Chlorosis .... 
Healthy 



27 Lymphadenoma 

35 Carbuncle 

38 Myxoma 

56 Healthy 

56 Osteo-arthritis . 

65 j Hernia 

90 Healthy . 



Total 



890 
957 
117 
334 
136 
994 
412 
365 
201 
338 
397 
801 
724 
390 



42 7,056 



155 
117 

155 

146 
168 
172 

163 
172 
120 
127 

186 



199 
212 

179 

201 
244 
193 

175 
225 
192 
165 
204 



Averag 



178 
159 
117 
167 
136 
166 
206 
182 
201 
169 
198 
160 
145 
195 



168 



APPENDIX I 



403 



TABLE III 

Cancer Cases 



A. Males- 



Name. 



Age. 



Locality of Disease. 




S=:5 
5 g ri 



O— o 

*5^ 



° h u 

MO & 



Doyle 32 

Mackie 32 

Rhead 34 

Ya Foo .... 44 

Nesborough 44 

Donahern . . 59 

Gardiner ... 59 

Welsh .... 65 

Whelan 68 



Stomach 5 699 

Lung (Secondary) . . 2 340 

Testicle 7 1,036 

Penis 7 1,201 

Lip 3 468 

Sigmoid 6 835 

Stomach 1 150 

Penis 6 1,013 

Tongue 5 928 

Total 42 6,670 



113 
129 
104 
132 
132 
106 

132 

140 



166 
211 

187 
197 
199 
175 

196 
234 



Averag 



140 
170 
148 
171 
156 
139 
150 
169 
186 



159 



B. Female s- 



Duncan 

McCann 

Evans . 

McQuillian 

Griffiths 

Jones . 

Hiles . . 

Walker 

Griffiths 

Griffiths 

Roberts 

Hall .. 

Cunning 



35 Stomach 

41 Liver 

42 Breast 

42 Uterus 

45 Breast and Pelvis 

45 Breast 

49 Breast 

54 Stomach 

56 Cervix Uteri . . . 

56 Breast 

56 Stomach | 3 

56 Breast 

66 Breast 

Total 



357 
323 
144 
217 
1,201 
119 



156 201 
121 202 



163 273 



178 
161 
144 
217 
200 
119 



2 


313 


146 


167 


156 


-5 


767 


147 


158 153 


5 


953 


121 


247 191 


7 


1,046 


109 


197 150 


3 


387 


96 


159 I 129 


1 


120 




... 120 


6 


804 


88 


158 


134 


42 


6,751 




Averag 


el61 



404 



APPENDIX I 



TABLE IV 

Average Number of Granules in (A) Largest, and (B) 
Smallest Cells 

1. Males and Females separated — 



Controls. Controls, j Cancer. 

Males. Females. Males. 

Table I. Table II. I Table III. 



Cancer. 
Females. 
Table III. 



(A) Average number of 
granules in largest cells 

(B) Average number of 
granules in smallest cells 




196 
127 



2. Males and Females combined- 





Average Number of 
Granules in Largest Cells. 


Average Number of 
Granules in Smallest Cells. 


Controls 

Cancer 


202 

196 


151 
126 







It should be noted that the greatest difference between Cancer 
and Control cells is in the smallest leucocytes. 



APPENDIX I 



405 



SUMMARY 



Total number of persons examined 69 

Total number of cells photographed 235 

Total number of granules counted 38,759 

Table showing differences between the cells of Control {healthy and diseases 
other than cancer) persons and Cancer persons 





Persons 
Examined. 


Cells j Granules 
Photographed. Counted. 


Average 
Granules 
per Cell. 


Controls 

Cancer 


47 
22 


151 

84 


25,338 
13,421 


168 
160 



Males- 



Table showing Influence of Sex 





Persons 
Examined. 


Cells 
Photographed. 


Granules 
Counted. 


Average 
Granules 
per Cell. 


Controls 

Cancer 


33 

9 


109 
42 


18,282 
6,670 


168 
159 



Females- 




Number of granules in smallest cell, 82. Number in largest cell, 287. 

Variation in the number of granules contained in the cells of 
one person 

21 cells from Cropper. Smallest cell contained 114 granules; 
largest contained 260. The average number of granules in the 21 
cells is 161. 



APPENDIX II ] 



SOME COMPARATIVE MEASUREMENTS OF THE LIVES OF LEUCO- 
CYTES 2 WHEN THE CELLS ARE RESTING IN THE PLASMATA 
OF DIFFERENT PERSONS 



AND THE POSSIBLE APPLICATION OF SUCH MEASUREMENTS AS AN AID TO 
DIAGNOSIS IN INFECTIVE DISEASE 

Of recent years I have been endeavouring to ascertain the effect 
produced by one person's plasma on the life of another person's 
leucocytes. It appeared reasonable to suppose that the plasma of 
a person suffering from an infective disease would be poisonous to 
the leucocytes of healthy persons. If this is the case it might also 
be reasonable to suppose that the same plasma would not be so 
poisonous to the leucocytes of another person suffering from the 
same disease, because it is probable that the cells would be already 
used to, or immune against, the toxin; and furthermore that if the 
toxin of one infective disease differs from the toxin of another 
infective disease, it might be inferred that an immunity on the 
part of a leucocyte against one disease will not render it immune 
against another. Therefore, provided it is possible to tell accurately 
when a leucocyte is dead — that is, if one can differentiate a living 

1 A method for estimating the number of living and dead leucocytes con- 
tained in a given sample of blood ; and another convenient formula for the 
preparation of "kinetic jelly." Being a paper reprinted from The Lancet of 
February 6, 1909, by kind permission of the and Editor of that Journal. 

2 The word "leucocyte" refers to the neutrophile polymorphonuclear 
leucocyte. 



406 



APPENDIX II 407 

from a dead cell — it also will become possible to measure the 
lengths of the lives of leucocytes after they have been removed 
from the body. And this will enable us to make comparative 
measurements of the lives of leucocytes when thev are mixed with 
the plasmata of different persons. Supposing, therefore, it is true 
that an infected plasma shortens the lives of a healthy person's 
leucocytes but does not shorten the lives of the leucocytes of another 
person suffering from the same disease, it may be useful to reverse 
the process and assist in the diagnosis of infective disease by making 
measurements of the lives of such a patient's leucocytes when they 
are mixed with dfferent plasmata. For instance, if the leucocytes 
of a person suffering from an indefinite infective disease are found 
to be easily killed by the plasmata of persons suffering from a 
variety of diseases, but are not comparatively easily killed by the 
pasma of a person suffering from, say, typhoid fever, it might be 
inferred that the patient is suffering from, or has recently suffered 
from, typhoid fever, because his leucocytes are used to, or immune 
against, that disease. 

The above is the enunciation of a problem which I set myself 
to solve several years ago, and this paper describes the experiments 
which have been conducted to investigate the last part of it — i.e. 
with the object of determining the actual measurements of the lives 
of leucocytes when they are placed in the plasmata of people who 
are suffering from various diseases. The earlier researches made 
in order to differentiate living from dead leucocytes have already 
been published in the Journal of Physiology (l), 1 and the actual 
method employed to estimate how many living and how many 
dead cells there may be in a given volume of citrated blood has been 
described in I lie Lancet of January 16, 1909 (2). This method 
may be again briefly summarised thus: 

Method for counting the number of living and dead leucocytes in 
a given sample of citrated blood. — The following solutions are pre- 
pared and a jelly is made from them. 1. A volume of Unna's 
polychrome methylene blue (Grtibler) is diluted with two volumes 
of water. 2. A solution containing 2 per cent of agar in water, 

1 The figures within parentheses refer to the bibliography at the end of the 
article. 



408 APPENDIX II 

filtered and sterilised. 3. An accurately neutralised solution con- 
taining 4 . 5 per cent sodium citrate, 1 . 5 per cent sodium chloride, 
and 0.225 per cent atropine sulphate. 4. A 5-per-cent solution 
of sodium bicarbonate. In a test-tube mix one cubic centimetre 
of the diluted stain, two cubic centimetres of the citrate solution, 
and three cubic centimetres of the molten agar solution. To 
this mixture a quantity of the alkaline sodium bicarbonate 
solution must be added in order to cause the excitant for leucocytes 
contained in the jelly to diffuse into the cells, and the quantity 
added varies with the temperature of the room. 1 If measurements 
are going to be made in a room with a temperature of between 
60° and 70° F., about . 25 cubic centimetre of the alkaline solution 
should be added. The mixture is then boiled until it froths up 
the tube and a drop poured on to a slide and allowed to set so as to 
form a film. Supposing a given capillary tube contains the blood- 
corpuscles of one person mixed with the plasma of another, the 
average number of living and dead leucocytes in the tube can be 
estimated by placing a drop of its contents on a cover-glass which 
is inverted and allowed to fall on the agar film. After two or 
three minutes the granules but not the nuclei of the living leuco- 
cytes will stain and those cells will show exaggerated amoeboid 
movements, whereas the dead cells will remain immobile. More- 
over, the dead cells may be achromatic (3), in which case they will 
not stain. Their nuclei may appear as a single nuclear mass, 
or their nuclei may even stain, or the dead cells may have under- 
gone other changes which have been described in former papers 
(1, 2). Field after field should be rapidly passed in front of 
a l-6th inch or equivalent objective and the number of the 
living and dead cells counted. Several preparations can be rapidly 
examined and an average struck so as to give an estimate of the 
number of living and dead cells in the given capillary tube. No 
difficulty is met with in making the counts, for living can be 
readily differentiated from dead cells by the presence or absence of 
exaggerated movements. 

If all the leucocytes appear to be dead, and especially if the 
agar jelly has not previously been tested, it is as well to control 
1 A scale has been given in the former paper. 



APPENDIX II 409 

the measurement — that is, to see that the jelly will actually excite 
living cells — by placing a drop of fresh citrated blood on to another 
part of the same film and noting whether stimulated movements of 
all the leucocytes occur. 

Procedure for the preparation of capillary tubes containing the 
plasma of one person and the leucocytes of another. — It will simplify 
description if the details of sterilisation and the precautions for 
ensuring asepsis are omitted. Since the presence of bacteria 
shortens the lives of leucocytes (2) it is obvious that aseptic pre- 
cautions are essential, but the details for sterilisation are so well 
known that they need hardly be repeated. A capillary tube of 
glass is prepared which has such a diameter that blood will run 
into it by capillarity and at the same time its flow can be controlled 
by gravity. I use a tube with a lumen of about two millimetres. 
15 portions equal to each other are marked off with a pencil. The 
marks begin at one end of the tube which is zero, but the tube 
is at least two inches longer than mark 15. The portions are 
rendered equal by calibration with mercury, and although the 
length of each is immaterial, I have found that about half a centi- 
metre is a convenient length for practical purposes and I use a tube 
about 13 centimeters long. A neutral solution is made which 
contains 3 per cent of sodium citrate and 1 per cent of sodium 
chloride. Some of this is drawn up into the tube until its upper 
limit or meniscus stands at mark 6. Blood from the finger of the 
person whose plasma is going to be tested is added until the 
meniscus stands at mark 12, care being taken that no bubble of 
air separates the two fluids. Mixture is carried out by allowing 
the two fluids to gravitate up and down the tube six times. The 
tube is sealed and centrifugalised; the blood being driven towards 
zero. The end remote from zero is then unsealed and the portion 
containing the precipitated corpuscles is separated and discarded 
by cutting the tube at 4. Eight portions of the tube now contain 
citrated plasma. If, owing to the sealing process, much of the tube 
has been occluded at zero the upper meniscus may stand above 
mark 12. This can be corrected by tapping out the excess of fluid 
on to a sterile slide, controlling the amount removed by the finger on 



410 APPENDIX II 

the end remote from the mark 4. The lower meniscus standing at 
4 where the tube has been cut, and the upper meniscus standing at 
12, blood from the finger of the persons whose corpuscles are going 
to be tested is added until the upper meniscus stands at 13 (i.e. the 
mixture equals 1-9). Mixture is ensured as before and the tube 
sealed. It will be seen that although the tube contains the plasma 
of both persons the corpuscles are bathed in a solution containing 
four times as much plasma of the first person as of the second. A 
series of tubes may thus be made. 

Appliance to ensure continual mixture and to prevent the cor- 
puscles from adhering to the glass. — If a capillary tube prepared 
in the way which has been described is laid on a flat surface, the 
corpuscles will soon gravitate to the most dependent side and will 
ultimately adhere to the glass. The following appliance prevents 
this. By means of a simple clockwork movement a split drum is 
made to revolve once in about three minutes. The drum is so 
adapted thaT the mouth of a long test-tube (having a diameter of 
one centimetre and the cavity of which is lined with a roll of blot- 
ting paper) fits accurately on to it and revolves with it. The 
apparatus is so arranged that the tube is horizontal and is of such 
a size that it can be placed in the incubator if necessary. The 
capillary tubes inserted into the test-tube are continually tumbling 
over each other by gravity as the test-tube revolves, and in so doing 
revolve themselves. The blood-cells in their turn are continually 
gravitating in different directions through the citrated plasmata. 
It has been found that this device prevents them adhering to the 
glass and ensures them being evenly distributed through the 
citrated plasmata provided the ends of the capillary tubes are not 
bent over when sealed. This apparatus also insures all capillary 
tubes being subjected to the same conditions of temperature. 

Procedure for measuring the lives of the leucocytes contained in 
the tubes. — Samples of the contents of the capillary tubes are 
examined on stimulating agar by the method already described. 
If all the cells are alive the tubes are resealed and returned to the 
revolving apparatus to be examined later, and so on. By this 
means the percentage of living and dead cells in a tube can be 



APPENDIX II 411 

estimated. It is important to remember that in striking these 
averages only an approximate estimate can be obtained, and that 
therefore the greater the number of tubes made the better, as the 
error decreases with the greater number of leucocytes counted. In 
the experiments which I am about to record I have counted about 
500 leucocytes in each case by making five films from each of five 
tubes, and counting about 20 leucocytes in each film. Since it is 
obvious that the greatest error may occur when the number of 
living approximates the number of dead cells in a tube, the follow- 
ing experiments would appear not to be very erroneous, judging by 
the application of Poisson's formula, which shows that supposing 
there are half a million leucocytes in the five tubes, which is an 
excessive estimate, a count of 500 cells would give a possible error 
of not more than about 6 per cent, even when the numbers 
approximate. 

Before enumerating the actual measurements there is yet 
another question to be considered, a point upon which I wish 
to lay great emphasis — namely, that all measurements of the 
lives of leucocytes should necessarily be comparative. For instance, 
it would be fallacious to say that a typhoid plasma killed a person's 
leucocytes more rapidly than a septicemic patient's plasma, when 
the typhoid measurement was made to-day and the septicemic 
measurement made three davs ago; for even if there was a great 
difference in the length of the lives and the same person's leucocytes 
were used, one cannot say that that person's leucocytes were in the 
same state to-day as they were three days ago, although the person 
is apparently in the same healthy condition. 

x\gain, I have shown (4) that the factor heat in accelerating 
the diffusion of substances into cells also materially affects the 
lives of the leucocytes, since the cells are necessarily resting in a 
citrate solution which is itself poisonous to some extent, and even 
the temperature of incubators is variable. It is thus of the utmost 
importance that when the lives of a person's leucocytes, which have 
been placed in the plasma of a person suffering from an infective 
disease, are measured, a simultaneous measurement of the same 
leucocytes shed at the same time must be made in the plasma of 
a healthy person. And it is only by the difference between the 



412 APPENDIX II 

two that the result can be determined. In other words, all 
measurements must be simultaneously controlled by other measure- 
ments and the contrast is the result. It is also obvious that since 
heat and the citrate solution both affect the lives of the cells, all 
tubes, whether containing infected or control plasma, must be 
subjected to the same conditions as regards temperature. And it 
is essential that the same citrate solution must be employed both 
for the test and the control. Unless these essential details are 
adhered to, any measurements may be considered to be worthless. 
Leucocytes are very sensitive to changes in temperature when they 
are resting in citrate solution, but if a change occurs and all tubes 
are subjected to the same change the contrast in the length of life 
holds good. The most favourable arrangement of the citrate 
solution has already been given. It should be quite neutral, 
because if alkaline it shortens the lives of the cells. 

Leucocytes appear to live longest at about 20° C. They will 
not live very long at 37°, and at 10° will live longer than at 37° but 
not so long as at 20° C. I have already suggested (4) that this 
may be due to the accelerated absorption of the poisonous salts in 
the citrate solution caused by heat, and this will also explain the 
early death in the presence of alkali which also accelerates diffusion. 
I presume that the reason why they live longer at 20° than at 10° 
is because their normal temperature is about 37° C. and that they 
die in the cold in spite of the delayed absorption. 

In the following experiments a temperature of 30° C. was em- 
ployed with the specified citrate solution, and control experiments 
were conducted in each case, the results given being the difference 
in the measurements between the test and control. 

Measurements 

Length of the life of healthy person's leucocytes when resting in 
their own plasma. — As has been shown in a former paper (2), an 
average shows that all the cells are alive in 24 hours; the majority 
are alive in 36 hours; about 50 per cent are dead in 48 hours; 
and all are dead in 86 hours. 

Healthy person's leucocytes; other healthy person's plasma.— All 
cells were alive in 14 hours; about 50 per cent were dead in 18 



APPENDIX II 413 

hours; the majority were dead in 22 hours; and all were usually 
dead in 28 hours. The difference between these averages may be 
said to be about 30 hours. I conclude that the plasma of one 
person is poisonous to another person's leucocytes. 

Healthy person's leucocytes; plasma from cases of typhoid fever. 
— All cells dead in 14 hours. Difference between test and control 
about six hours, which is the average out of four cases. 

Healthy person's leucocytes; plasma from cases of malaria. — 
Majority of cells dead in 16 hours, a few alive in 18 hours. 
Occasionally 50 per cent were alive in 16 hours. Average differ- 
ence between 12 cases and their controls about two hours. 

Healthy person's leucocytes; plasma from cases of phthisis. — 
Majority dead in 17 hours. Average difference between five 
cases and their controls about one hour. Sometimes it was as 
much as four hours, but in very chronic cases there was little 
difference. 

Healthy person's leucocytes; plasma from a case of osteo-myelitis. 
— 50 per cent dead in 14 hours. Repeated with a case of gan- 
genous appendicitis the films showed that the majority were 
dead in 14 hours. The difference between these cases and their 
controls were five hours and three and three-quarter hours 
respectively. 

Healthy person's leucocytes; plasma from a case of purpura 
hemorrhagica. — Majority dead in 15 hours; all dead in 20 hours. 
Difference from controls five hours. 

Healthy person's leucocytes; plasma from a case of chorea. — All 
cells dead in 14 hours. Difference about six hours. 

Leucocytes from cases of typhoid fever; plasma from other cases 
of typhoid fever. — Average from three groups of cases, all of which 
reacted to Widal's reaction and were in the third or fourth week 
of the disease except one which was convalescent. These groups 
include the cases mentioned above. There was never a difference 
of more than one and a half hours between the death of the 
majority of cells in test and control tubes. 

Leucocytes from cases of malaria; plasma from other cases of 
malaria. — Five cases. The majority of cells in all cases were alive 
in 18 hours. Practically no difference from controls. 



414 APPENDIX II 

Leucocytes from cases of phthisis; plasma from other cases of 
phthisis. — Four experiments. 50 per cent dead was the average 
in 18 hours; very little difference from controls, sometimes the 
cells lived longer than in the controls. 

Leucocytes from cases of malaria; plasma from cases of typhoid 
fever. — The majority of the cells in most instances were dead in 
14 hours. Differences varied from four to six hours. 

Leucocytes from cases of typhoid fever; plasma from cases of 
malaria. — About 50 per cent were usually dead in 16 hours and 
all were dead in 20 hours in all cases. Five cases tried; average 
difference about three hours. 

Healthy person's leucocytes; plasma from cases of carcinoma. — 
Seven cases; all cells alive in 16 hours; a large number alive in 20 
hours. Usually there was little difference between the effect of 
cancer plasma and that of a healthy person. 

From the foregoing measurements it would appear that in the 
cases which have been experimented with the plasma of persons 
suffering from infective diseases is poisonous to a healthy person's 
leucocytes and to the leucocytes of another person suffering from 
another disease, but is not so poisonous to the leucocytes of another 
person suffering from the same disease. I submit that it may be 
reasonable to suppose that such may be the case in other infective 
diseases. 

Precautions. — In comparing the lengths of the lives of leucocytes 
of persons suffering from chronic infective diseases both in another 
infected person's plasma and in healthy plasma, I have frequently 
found that such cells will not live so long as the cells of healthy 
persons subjected to the same conditions. This was further inves- 
tigated by comparing the lives of leucocytes taken from cases of 
chronic illnesses in their own plasmata with the length of the lives 
of the cells of healthy persons in their own healthy plasmata. In 
cases of chronic phthisis, malaria, Hodgkin's disease, 1 etc., I have 
found that the leucocytes will not live even in their own plasma 
nearly so long as if they belonged to a healthy person, as much as 

1 It has been noticed that stain will diffuse more readily into the blood- 
cells of these patients — that is, that these diseases, and probably other 
chronic illnesses, cause a lowered "coefficient of diffusion" in blood-cor- 
puscles. 



APPENDIX II 415 

a day's difference having been observed; and we may infer that 
these diseases, and probably others also, cause a loss of vitality in 
the patient's leucocytes, so that by this procedure the loss of vitality 
can be measured. It is important to remember this point, for if 
the making of a measurement is delayed it may be found that all 
the cells are dead in both control and. test preparations. This 
method of measuring the lives of leucocytes may also prove of 
value in prognosis as well as in diagnosis. 

I do not think that any difficulty will be met with in making 
the counts, with the exception of a possible one caused by the 
agglutination of the leucocytes. Occasionally large clumps are met 
with. If the cells are clumped, however, it does not necessarily 
follow that they are dead — far from it, for they may be very active, 
though I am of opinion that if clumped death will soon occur. 
The cells in a clump can generally be counted. Ruptured cells 
are counted as dead. If bacteria are seen in large numbers in a 
film the capillary tube is discarded. The revolving apparatus is 
not essential, but more constant results have been obtained by its 
use. As far as possible I have purposely avoided handling the 
blood of the person whose leucocytes are to be tested, for fear of 
injuring the cells. The variations of the alkalinity of the plasma 
may, I think, be neglected, as it is not sufficient materially to alter 
the length of the lives of the cells. This is borne out by the 
experiments with cancer plasma, because that plasma is more alka- 
line than normal and yet does not shorten life. 

Summary 

I fear that it is too early to arrive at any definite conclusions 
from so small a number of experiments, but I think that there 
publication is justified in order to explain the method employed 
and because the results are sufficiently promising to warrant further 
investigation, though the work must still be regarded as being in 
the experimental stage. I hope that this method will be tried by 
others, as the problem given in the enunciation may lead to im- 
portant developments, and especially as this kind of research in- 
volves the striking of averages and a large amount of experiment to 



416 APPENDIX II 

determine the points. The method may also be useful to others 
studying other branches of immunity. As I have already stated, 
my aim is to be able to assist in the diagnosis of infective disease 
by this method, but a large amount of material will be required 
before one can determine its value in this direction, and I have 
mentioned its possibilities with reference to prognosis. The stage 
in a disease in which measurable immunity appears in a leucocyte 
also remains to be determined. 

To summarise the method by which I endeavour to assist in 
a diagnosis in a case of infective disease, a small quantity of blood 
from a patient is mixed with eight times its volume of the citrated 
plasma of other persons who are known to be suffering from certain 
infective diseases and also with the citrated plasma of a healthy 
person. For this last purpose I sometimes use my own plasma. 
The method has been described. The capillary tubes are kept 
together in the revolving apparatus for about 14 hours. Then 
some agar films are prepared from jelly which will excite move- 
ments in living leucocytes, and samples of the contents of the tubes 
are examined on these films. The number of living and dead cells 
are averaged, and the difference between the lengths of the lives of 
the cells when resting in healthy and infected plasmata are deter- 
mined. When an infected plasma is found which will not com- 
paratively shorten the lives of the patient's leucocytes, it seems 
probable that the patient is suffering from the same disease as the 
person from whom the plasma was taken. I generally confirm 
this procedure by reversing the process and trying the patient's 
plasma on the leucocytes of other persons suffering from the dis- 
ease determined, taking care to make controls in this case as well 
as in the first by making measurements with healthy plasma and 
with the plasma of persons suffering from other diseases. 

The method described in this paper has two disadvantages: 
first, in keeping the tubes at 30° C, and, secondly, in counting 
500 leucocytes in each case, which is most tedious. The rest of 
the method takes very little time; collecting the plasmata and 
mixing them with the patient's corpuscles is soon accomplished, 
and when the tubes are in the revolving apparatus they require no 
further attention until the time has come to estimate the number 



APPENDIX II 417 

of living and dead cells in them. The agar jelly can be made from 
stock solutions as specified and kept in test-tubes for months, as 
moulds will not grow on it. Films are rapidly prepared by boiling 
the jelly in a tube in a spirit-lamp flame. 

With regard to the two disadvantages, an incubator working 
at 30° C. is not usually within reach even in laboratories, although 
Hearson's apparatus will maintain this temperature if fitted with 
a special capsule. Since my aim is to make this possible diagnos- 
tic method suitable for practical purposes even away from the vicin- 
ity of a laboratory, I dispense with an incubator and employ the 
ordinary temperature of a room, say between 60° and 70° F. In 
order to do this the citrate solution is modified. If the solution 
already specified were used at such a temperature the leucocytes 
might live for a long time even in an infected plasma, and a day 
or two might elapse before sufficient deaths occurred among the 
cells to make a contrast. Consequently I deliberately shorten the 
life of the cells by using a solution containing 1 . 2-per-cent sodium 
citrate and 1-per-cent sodium chloride. As the same solution is 
employed for all tubes the artificial shortening of life does not 
appear to vitiate the results. There are several ways by which 
this shortening of life can be accomplished, though I consider the 
lowering of the sodium citrate content to be the most suitable. 
Using this solution it has been found that the majority of healthy 
cells in another healthy person's plasma are dead in about 24 hours 
if kept at the room temperature, which, of course, may be variable. 
So a contrast can usually be obtained within 24 hours of mixing 
the bloods. With regard to the second disadvantage, I hope by 
experiment to ascertain the minimum number of leucocytes which 
it may be necessary to count to obtain a trustworthy average. I 
am sure that a smaller number than 500 will be sufficient. I am 
also experimenting with a greater concentration of plasma with a 
view to obtaining a wider contrast between the length of the 
lives of cells in healthy and infected plasma. 

In conclusion, I wish to suggest that this method may also be 
useful from a medico-legal aspect, for I have found the leucocytes 
alive in the blood removed from the hearts of bodies which have 
been lying in the mortuary for 24 hours or more, and it may be 

27 



418 APPENDIX II 

possible to state how long a person has been dead by estimating 
the percentage of living cells so many hours after the death of the 
subject. 

Bibliography. — H. C. Ross: (1) " On the Death of Leucocytes," Journal of 
Physiology, vol. xxxvii., 1908, p. 327; (2) "On a Combination of Substances 
which Excites Amoeboid Movements in Leucocytes," The Lancet, Jan. 16, 
1909, p. 152; (3) "On the Cause of Achromasia in Leucocytes," The Lancet, 
Jan. 23, 1909, p. 226; (4) "On the Modification of the Excitant for Leu- 
cocytes composed of Methylene Blue and Atropine," The Lancet, Jan. 30, 
1909, p. 313. 



APPENDIX III 

A METHOD BY WHICH CELLS CAN BE EXAMINED MICROSCOPICALLY 
BETWEEN A COVER-GLASS AND A JELLY-FILM WITHOUT 
THE FORMER EXERTING ANY PRESSURE ON THEM. (a 

"hanging drop" PREPARATION WITH THE JELLY method) 

Two round cover-glasses are required. One should have a diame- 
ter of half an inch, the other of seven-eighths of an inch. The 
jelly from which the film is to be prepared is boiled and a drop 
of it run on to a slide. Immediately, before the jelly has had 
time to set, the small cover-glass is allowed to fall flat on the 
centre of the jelly-film on the slide. Since the jelly is not set, 
the cover-glass sinks into but not actually through it. The film 
with the cover-glass embedded in it is allowed to set for about five 
minutes. One needle is then placed vertically against one edge 
of the small cover-glass embedded in the jelly, and the point of 
another needle is inserted under the opposite edge of the cover- 
glass. By a jerk of this needle the embedded cover-glass is lifted 
out of the jelly, when it will be found that a shallow circular 
depression exactly corresponding to the cover-glass is left in the 
jelly-film. The base and sides of the depression will, of course, 
be composed of jelly. The cells to be examined are placed in 
citrate solution on the large cover-glass, which is inverted and 
allowed to fall flat over the depression in the film. By this 
means the large cover-glass is resting on the raised sides of the 
depression, but the cells are in the depression. They can now 
be made to absorb substances from the jelly, but the cover-glass 
does not press them into it unless the cells are very large. This 
method is useful for the in-vitro staining of motile bacteria, try- 
panosomes, etc. 

419 



APPENDIX IV 

A POSSIBLE ASSOCIATION BETWEEN THE AUXETICS OF 
HEALING AND IMMUNITY AGAINST INFECTIVE 
DISEASE 

The fact that auxetics contained in the remains of dead tissues 
and in globin will cause the cell-proliferation of healing has 
suggested a new line of research connected with the problem of 
immunity. Since the cell-proliferation of healing is caused by 
chemical agents, and since the actions of these agents can be 
augmented by substances produced by bacteria, and inhibited 
by normal serum, it may be useful to ascertain the action of 
disease germs on (1) the remains of dead tissues and globin, and the 
auxetic it contains, and (2) on serum. It is obvious that if disease- 
germs decompose auxetics, there will be less cell-proliferation of 
healing; but if they produce substances which augment the action 
of auxetics, or if they prevent the inhibitory action of serum, then 
they will tend to assist in healing an injury. Before any disease- 
germ can obtain a footing in the body it must cause an injury 
which is followed by an attempt at healing. If this healing is 
prevented, disease will be the result. If, however, healing occurs 
successfully, the patient will remain immune. Hence this sugges- 
that the action of disease-germs on the sources of the causes of the 
cell-proliferation of healing should be investigated. In reality, 
the problem is a bacteriological one, but the investigation of it will 
not, I think, be very difficult. 



420 



INDEX. 



Achromasia, 4, 14, 21, 33, 43, 55 

Acids, 71, 89 

Aconitine, 149 

Agar, 7, 9, 36, 40, 83 

Alkalies, 71, 77, 86, 88 

Alkaloids, 149, 157, 352 

Amitotic divisions, 297 

Amceba Coli, 98, 400 

Amoeboid movements, 67 

Apparatus, 18, 20, 27, 34, 410 

Archoplasm, 112, 123 

Artefacts, 17 

Asymmetrical mitosis, 10, 12, 44, 

132, 235, 240, 300 
Augmentation of cell-division, 227, 

310 
Auxetics, 6, 232, 292 
Azur dye, 2, 237, 347 

Bacillus subtilis, 366 
Bacterial toxins, 8 
Basophile leucocyte, 96, 274, 281 
Benign tumours, 7, 339, 340 
Blood alkalinity in cancer, 176 
Blood-platelets, 5, 102, 113, 117, 124 
Blood-serum, 9, 374, 376 
Brownian movements, 38, 39, 52 
Brucine, 149 

Cadaverine, 350, 365 

Callus, 336 

Cancer, aetiology of, 161, 164, 360 

age-incidence of, 161, 360 

cases of, 379 

causes of (theory), 360 

climatic incidence of, 164, 371 

place-incidence of, 371 

plasma, 159 

prevention of recurrence of, 395 

recurrence of, 397 

site-incidence of, 363 
Capillary tubes, 18 
Centrosomes of leucocytes, 258 

of lymphocytes, 186 



Chemotaxis, 150 

Choline, 352, 365 

Chorea, 8 

Chromosomes of leucocytes, 258 

number of, 239 

of lymphocytes, 186 
Citrate solution, 41, 82 
Citric acid, 85, 88 
Cocaine, 149 
Codeine, 149 

Coefficient jelly, 85, et seq. 
Coefficient of diffusion, 61, et seq. 
Concentration of substances, 68 
Cornea, 337 
"Corns," 337 
Crenation, 38 
Crescent, malarial, 323 
Cytogeny, causes of, 158, 166 
Cytoplasm, 52, 59, 62, 82 

Death, delay of, 107 

in cancer, 165, 366 
Death-struggles, 147, 246, 346 
Decrepitude, 163 
Defibrinated blood, 378 
Diffusion of substances, 67, et seq. 

of two substances, 145 

vacuoles, 103, 111 
Doses of alkaloids, 148 
Drugs, 61, 63 

Elimination, 338 

Embryo, 7 

Eosinophile granules, 274, 281, 401 

leucocytes, 94, 293 
Epithelial cells, 73, 352 
Erythrocytes, 72 
Examination of specimen, 102 
Excess of diffusion, 103 
Excitation, 130 

" Experimental ten minutes," 249 
Extracts, 7, 298, 300, 315 



Fertilisation, 167, 339 



421 



422 



INDEX. 



Fibroids of uterus, 340 
"Flagellation," 175, 272 

of malaria parasite, 323 
Focusing, 33 

Gametophytic tissue, 178 
Globin, 322, 342 
Granulation tissue, 338, 341 
Granules, Altmann's, 52, et seq.. 223 
counting, 273, 284 

Haemal glands, 180, 183 
Hsematin, 324, 331 
Haemoglobin, 128, 322, 397 
Haemolysins, 38, 98 
"Hanging drop." 150, 419 
Healing, 41, 168, 176, 319 
Heat, 70, 78, 88 
Hyaline cell, 4, 60 
Hydrocyanic acid, 132, 148 

Immunity, 9, 420 

Incubator, 94, 98 

Index of diffusion, 82, 94 

Injury, 41 

Irritation, 166, 170, 337, 348 

Karyokinesis, 6 
Katabolism, 361 
Kinetic jelly, 133, 377, 407 
Kreatin, 316 

Lantern slides, 14 
Leucocytes, Altmann's granules of, 
256 

bursting of, 107 

divisions of, 252 
Leukaemia, 175, 398 
Life, climax of, 162 
Lives of leucocytes, 132, 406 
Lymphadenoma, 351 
Lymphocytes in cancer, 393 
Lymphocytes, mitosis of, 186 

Maiotic divisions, 177, 239 
Melanin, 322, 324 
Malaria parasite, 323, 331 
Malignant proliferation, 12 
Methylene blue, 3 
Metastasis, 165, 367 
Mitosis, 185, 252 
Morphine, 40, 118, 149 
Moulds, 85 
Mutations, 369 

Neutral point, 89 

red, 3 
Nitro-benzol, 132, 148 



Nuclei, lobes of, 5, 10, 43 
Nuclein, 178 

Opsonins, 130, 156 
Osmosis, 59, 129 
Osteo-arthritis, 163 
Ova, 176 
Oxygen, 148 

Pancreas, 364 
Phagocytosis, 155 
Photomicrography, 3, 14, 17 
Pigmentation, 322 
Pilocarpine, 149 
Plimmer's bodies, 181 
Poisons, 8, 64 
Potassium oxalate, 39 
Precautions, 99 
Protoplasm, 64, 69 
Pseudopodia, 51, 56, 134 
Putrefaction, 300, 350, 366 
Pyridine, 149 

Quinine, 149 
Quinoline, 150 

Red cells, 82, 117, 127, 294 
"Red spots," 103 
Reduction divisions, 239, 271 
Reproduction divisions, 239, 271 
Reproductive cells, 240 
Resting-stage, 97 
Rheumatism, 8 

Salts, 37, 59, 86, 97 
Sarcoma, 161, 397 

cell-granules in, 291 

melanotic, 322 
Serum, 66, 374 
Sexual form of malaria parasite, 323 , 

331 
Sodium chloride, 38, 39, 78, 84, 88 
Sodium citrate, 38, 44, 66, 76, 78, 88. 
Somatic divisions, 239 
Spermatozoon, 167, 339 
Spindle, 12, 185, 257 
Spirochceta refringens, 97 
Staining, extent of, 75 
Strychnine, 149 
Suprarenal gland, 306, 345 
Syphilis, 164, 364 

Technique, for inducing mitosis, 245 
Time, a factor of diffusion, 69, 78, 88 
Tissue cells, 41 

Transplantation of tumours, 369 
Trepanema pallida, 364 



INDEX. 423 

Ulcers, 341, 345 Variables, equation of, 145 

malignant, 384 Vitality, 163, 363 
Units, 76, 88, 97 

Ur^99 Stain ' 43 ' ^ 68 ' 7? Wandering cells, 240 

Vacuoles, diffusion, 104 Xanthin, 316 

ordinary, 103 X-ray cancer, 170 



OEC 24 1^10 



One copy del. to Cat. Div. 



24 » w 



LIBRARY OF CONGRESS 



022 194 857 9 






